Note: Descriptions are shown in the official language in which they were submitted.
~ 94/03159 21~1 g 5 9 pcr/Au93/oo392
CONTROLIED RELEASE IMPLANTS
FTF.T n OF THF TNVFNTION
S This invention relates to imrl~ntc CQI~t~ g active ingrerlient~ especially drugs or
veterinary products suitable for ~l...i..i~l. ation to h1lm~nc and ~nim~lc in which the
active ingredient is required to be ~ ed in a pulsatile release profile.
Prior art imp1~ntc are typically of the reservoir type and usually co~t~in a single
10 active ingredient and provide the c~..li..i~ous release of the active in a zero or first
order mode of release kinetiec
Present metho~lc of m~king these prior art imp1~ntc use basically two approaches, viz,
(i) tabletting; and (ii) melt proceccin~. Re~ece..t~tives of these approaches are
15 described as follows:
(i) T~h1ettin~ (Tnt.orn~tion~l P~t~nt ~li-~tion No. P(~T/AIJ87/00139)
A water incolllhle ~oYririçnt (eg, r~ lm lJhos~.h~te) is thoroughly mixed with
a bioactive agent such as a ~rotei~ or pepti-le in an amount sllffieient to givethe required dosage unit of active ingredient in the final product. The
bioactive agent is usually in the form of a so1lltion or dispersion or powder
to f~ lit~te miYing. A water so1nhle ~Y~ipi~nt (eg, lactose), if used, is then
added, together with the other desired additives, eg, a lubricating agent such
as m~ s;~.~.. stearate, and ixed to form a homogeneous dry powder. The
powder is then co~ essed into a tablet of the desired size and shape. The
co~essed tablet is then coated in a pan coater by spraying with a solution
or dispersion of the ro~ting material in an amount sufficient to give impl~nt~
with the reguired co~ting thickn-occ
In an alternative coating method known as Wurster coating, the tablet is
coated in a flllirli7e~ bed system.
Wo 94/03159 PCr/AU93/00392 ~
2141459
2 -
(ii) Melt Pror~ccir~ ("A sllct~in~-l rele~ce Iverme~tin impl~nt for livestork pect
control" by J ~ll~n Miller, R ) nrllmmonrl n n C~ehler in "Contrnlled
Rel~ce 1 )~liv~ry ~yst~mc" ed. Theodore ~ Rncem~n ~n~ ~ 7. M~ncdorf, 1983,
M~rcel-nekk~r; Ch~pter 15, pp ??~-?.~6)
An imrl~nt cont~ining 20% Ivermectinwas form~ te-l by dissolving technic~l
Ivermectin in a melt of polyethylene glycol (PEC~) (MW 15,000 - 20,000).
The solution was then drawn by vacuum into a 3 mm internal diameter
Teflon tube and allowed to cool. Upon cooling the r~cl~lt~nt solid rod (3mm
~i~m~ter) was removed and trimmed to the desired weight of 400 mg.
Tmrl~nt products of this type are solid cylindrical rods of varying length and
diameter and can have shapes ranging from flat discs to fine needles. This
type of imrl~nt is useful where a prolonged c~ nllc supply of the drug is
required.
The pan co~ting and Wurster coating methn-lc involve sllhst~nti~l contact between
the imrl~ntc being coated. During the drying process, the coating gets sticky
reslllting in impl~ntc stirking together or lwi.~.;,.g". It has been attempted to
.~.r~e this problem by the ~rl~lition of additives and mo-lifi~rs in the coating.
20 When the impl~ntc are to be used for ~ dLion to hnm~nc or ~nim~lc, the
additives which may be used are limited to those a~ al~ 'eS cleared for regulatory
use. Polyester Cc~tin~c~ for toY~mrl~ have been cleared for re~ tory u e howeverthece ~lesen~ a particular problem in the pan coating method due to a&esion of the
partide_ during the drying process. In ~rlrlitinn pinholes and other discontinnititos
25 can for_ in the outer coat during the drying process.
It would be desirable to avoid mutual contact of the impl~ntc during the drying
process and to be able to control the drying process to avoid the formation of
pinhnles and ~ co~ ;es
There are freguent ci~ "~ n~es where impl~ntc are re~uired to provide a pulsatile
release of an active ingredient. An example of such circurnstances is the so-called
~ W O 94/031~9 214 I 4 5 9 PC~r/A U93/00392
"one shot vaccine" concept. V~c~in~tion has been used to protect hllm~n~ and
~nim~lc against bacterial and viral infectious ~ice~c~os. In the case of vaccines
prepared in the form of killed su~;~ions of bacteria or viruses, or in the form of
conjugated tnYni~ls, repeated injections at specific ti_e intervals are required in order
5 for the v~crin~tion to effect adequate levels of i"""""ological res~llse. These
intervals may typically range from a few weeks to several mont_s. Due to prevailing
epiclçmiological, social, ec~"o",ic ~ce-~ibility, human tc~lA.\~t nt~l~ or simply
collve,.ience reasons, it is highly desirable that effective protection against ~i~e~ces
can be obtained with single injectionc In order to make this possible the one shot
10 v~ n~s have to release the actives at the fc~ el intervals, i.e. in a pulsatile mode,
in the le~ ed profile, i.e. a "du~ ". A further applic ~tinn of the "one shot vaccine"
approach is a 6 month or 12 month contraceptive impl~nt or vaginal su~o~ito~
which delivers the contracc~ hormones in a sllcce~;Q.. of pulses, the active
ingredient in this applic~tinn not being a vaccine.
A further no.l ~accillc ~Y~mple relates to the need for delivery of reaction mi~ctures
to sites of action, specific~lly the delivery of the la~;~u~rn~ ce thiocyanate enzyme
substrate re~tion system to the hin~ t~ of piglets in the control of diarrhoea.
La~;~o~.r~ e, coupled with a peroxide ge~ ~a~i~g ny~ e (eg, x~nthine and
20 x;-..lhi~.r nYirl~ce) CO~ SCN to SCNO, a very l~a;live and lethal ion for micro-
Olg,...;~ . Pulse release terhnnlogy can be used to address the problem of
rCi~r~
Constructing a delivery vehicle to meet these sorts of functional requirements
25 tlem~nfls a te~ hnology of m ~king co. . .p~ - 1 -. .ent~liced structures. The manufacturing
process should ~,efelably be simpl~, v~ns~tile and ~m~n~hle to me( h~ni~tion and
~ llt-)m ~tic~n.
Although the most i ~ ~t attribute of a controlled release drug delivery device
30 is its capabilityto m~int~in a the.~ ;c~llyerr~ level of drug in an animal body
over a srhedllled period of time, its adoption nltim~tely depends on the cost,
co,lv~ n~e, and ease of its fabrication and ~.l...i..i~ation (1).
W O 94/03159 ~141 4 5 9 PC~r/A U93/00392
In terms of the ease and co..v.~..ien.-e of ~.~minictering these devices as imrl~ntc,
shapes like sheets, films or hPmicpheres are generally impractical. However rods,
nee~lles or cylinders are readily adapted for parenteral imrl~nt~tinn using a
co~vGnlional hypodermic nPelle.
Pro.~llring eccentric shapes such as rods, n.-e.~lP~ ~r cylinders with the abovedescribed prior art methods, is also problematic. The pan coating and Wurster
ro~tin~ methn ls are most ~mPn~hle to imrl~ntc of a generally rounded shape.
10 The applicant's cc,~l~-li.\g Appli.~tion PCI/AU93/00083 discloses a method ofmAkin~ imrl~ntc which are suitable for co..lillllous release of an active ingredient
over a period of time which consist of a body member co".p,~ing a membrane
forming a wall around a core matrix and co~l~ing material which is substantiallyiLIl~ViOUS to the active ingredient .-J~nt~inPc1 within the core matri~ The cylinder
15 is generally open ended, the active ingredient being rele~cecl directly through the
open ends of the cylinder. In order to make an impl~nt suitable for pulsatile release
using this terhni.lue, it is n~cpcc~ry to separately coat the open ends of the impl~nt
This s~ate co~ting step is ~ clumsy, and time co.,~lmin~
20 A need accoldi~ exists for a simple and econnmi. ~l m~nllf~.-tllring process for
imrl~ntc which can release bioactive materials and which can be readily adapted for
comrl~Y in particular, pulsatile release of these materials.
AccoL.liLIgly, one aspect of the ~ se.~t invention cont~mrl~tPc a pulse release
25 imrl~nt com~ illg:
an axial biodegradable core;
a first concentric layer co~ g dehydrated hydrogel cont~ining an active
ingredient; and
an outer co~ting, said outer coating being removable by the environment in
30 which the imrl~nt will reside after ~I",i~ , ation.
--WO 94J03159 41 4 ~ 9 pcr/Aus3/oo392
In a further embodiment of the present invention there is provided a process of
e~ lg a pulse-release impl~nt inrlnc~ing the steps of:
co~ting an axially tiicposecl biodegradable core material with a hydrogel
cont~ining an active ingredient to form a conrentric coating;
S se~a,a~ing the coating into discrete se menn disposed along the core
material;
dehy~ ~ing the h~l~ogel;
coating the discrete segm~ntc with an outer coating, said outer coating being
removable by the ellvilo~ nt in which the imrl~nt will reside after ~rllni~ tion;
and
removing the P~se~ core material to obtain the pulse release imp1~ntc
The biodegradable core may be formed from any suitable material. Pl ~ fel ably the
core is bioc~ tible. The core may be formecl from a string, suture or rod.
The term l~ vgel is uced in its ord~ art rec~p.-i~ed me~ninE of a watel-based
three ~lim~ncion~l nonflowable amvl~hvus structure. The gel maybe created by ionic
or hydrogen bond inter~innc. Hydrogels of particular mte.~ sl consist of a solution
of a pvlymer in water which under controllable CO~ C can be made to adopt
20 either a fluid or semi-solid ~nfi~lration. This allows the hydrogel to be applied as
a fluid to the core member and retain its shape as a semi-solid form. Methods toinduce the t~ ;ol- in~ e tell~ ul~ control or the use of cross-linking agents
such as t~ lm ions. The te~la~u~ control ,llelllncl is suitable for gels such asagar and gelatine and the cross-linking metho~l is sllit~ble for hydrogels formed from
25 substances such as ~l in~te polymers.
In the ~rese~lt invention, a hydrogel in a fluid form is applied to the axial
biodegradable core, a tr~ncition to a semi-solid state is in~ ec~ and the hydrogel is
thereby immobilised. In this state, the hy-drogel may be cut and otherwise
30 maniplll~tecl When water is removed from the semi-solid form, a dehydrated
hydrogel is form~.-l The dehy~ated hy~ogel forms a rigid solid which is suitablefor storage and h~n~11ing
WO 94/03159 21. 41 ~5 9 PCI/AU93/00392 ~
Any suitable hydrogel may be used. F~mrles are gel~tine, agar, ~lginates~
carrageenan, gum gr~g~ nth, acacia, or corn starch. It may also be desirable to
in~ le other components in the first concentric layer. F-~mrles are disintegrating
agents such as corn starch, potato starch, alginic acid and the like and/or a lubricant
S such as m~gnesillm stearate. Osmotic morlifi-ors such as sucrose and gl~1r~se may
also be desirable. All such CO~ CIlLS, sho~iltl be subst~nti~lly pharmaceutically
pure and non tc)x,c in the amounts employed and should be biocompatible and
compatible with the active ingredient when used for human or animal use.
10 The active ingredient is typically a bioactive mc~lecllle and incl-1-les any native,
synthetic or recombinant pharm~elltic~l agent or food additive or supplement
in~ 1llclin~ antigens, antibodies, cytokines, growth promotants, hormones, cancer cell
inhibitory moleclllP5 or agents, immllne stimlll~ntc or ~u~læc~ntc, anti-microbial
agents inchl~tin~ antibiotics, anti-viral agents, vit~minc, minerals or inorganic or
15 organic nutrient~s. The active ingredient may ~~ ~c one type of bioactive molecule
or may be a ~ ule of ~rr~e~ bioactive molecules. In a ~lefel~ed embodiment
the active ingredient inclll~tes antigen~s from the clostridial family.
The outer ro~tin~ may be formed of any suitable bior~mratible substance. The
20 outer co~ting is generally meml~r~ous or polymeric and is sllhst~ntizllly illl~Cl vious
to the active ingredient. The majority of the active material will be delivered or
r~le~ced as a result of the removal of the outer co~ting ~...r~;..g the hrst concentric
layer. F.~mple~s of suitable coating materials in~ e mo-iified starches, sugars, poly
anhydrides, polyorthoecters, bioerodible polyc:,lcl:, and the polylacticlpolyglycolic
25 acid f~mily of polymers. Polylactic/polyglycolic acids are particularly suitable as they
are widely commercially available v.~ith various degradation profiles and have
regulatory clearance.
The co~ting may have a thickness of from typically lO~n to l,OOO,um depending on30 the app1ic~tion of the impl~nt and the permeability or degradability of the coating.
o 94/03159 ~! ~ 414 5 9 Pcr/Au93/oo392
Sli~ling
The imrl~nt device may be in any suitable shape int lll~ine elongate, oval, round,
ball, capsule, rod, needle, or cylinder shape. Conveniently, the shape is an elongate
S cylindrical, rod or needle shape. In a most ~refe,lcd embodiment, the imrl~nt device
is elongate and generally ~cal.
In the ~lucess of the present hl~ .~ion, an axially ~lisposed biodegradable corematerial is coated with a hydrogel Cb..~i"i,.g an actIve ingredient to form a
10 concentric co~ting~ .~m~ r clis~te gel se~n~ontc~ which remain supported by the
biodegradable core can be created by cutting the outer concentric hydrogel layer in
such a way that the biodegradable core remains intact and sliding the cut segment
along the axially dis~osed biodegradable core so as to form a space between the
discrete gel se~n~ntc This operationwill hereinafter be refe.l~ d to as the "cut/slide
15 operation".
The cut/slide operation may be ~,.;. r~" ...cd after the hydrogel has been dried and
prior to co~tine hc.... ._r it is generally easier to ~.rulm the cut/slide operation on
wet hydrogel. Ther~ rwe in the ~ocess of the l,ieseilt invention the separation and
20 dchy~llation steps may take place in either order. For ey~mrle the hydrogel may
be dehydrated before se~ tion into discn te se~ ntc although it is preferred that
the hydrogel is s_~atcd before dch~l~aliolL
The outer co~*n~ may be ~rplied in any suitable m~nn-or. For ~Y~mple, the outer
25 coa*ng may be applied by means of a mould, dipping, s~. aji,lg or by application via
a "rod" or ' wic~".
The above cut/slide opera*on ù~reoll,es certain deffciencies of known prior art
co~tin~ methods in that the problems of the impl~ntc st~ ng together during the
30 drying ploeess iS U~r~llle and the problem of pinholes and disco.~ ies in the- outer coat is alleviated as the drying process of the prese~t inven*on may be better
controlled.
wo 94/03159 PCr/A~193/00392 ~
2~4145~ '
- 8
Once the outer coating has been applied, the exposed core material may be cut away
rçsllltin~ in discrete pulse release impi~ntc.
The process of the present invention is readily adaptable for incorporation of heat
S labile active ingreriisntc. It pl~esen~ few COllaLldiL~tS wlth respect to the choice of
active ingredients; and it should be noted that by usl~g a biodegradable structural
:~iU~ ul l, such as surgical suture, the central core does not need to be removed from
the final imrl~nt product.
10 The present device also provides the basis on which further r~fin~m~nt or
sophistil ~tinn of release can be effected. For in~n~ e by usirlg as the hydrogel layer
bioerodible polyorthoester polymerc prepared by the reaction bet~veen 3,9-
bis(ethylidene-2,4,8,10-tetrau~iro [s~s]-lln~lec~ne) and various ratios of trans-
cyclohto~neclimethanol and 1~6-hey~n~linl (2), the production method can be readily
15 adopted for mass production of needle injectable imrl~ntc of ~,.til",nour agents such
as 5 flu~o-~l acil for release in a time independent mode. Likewise, by using collagen
poly (HEMA) ~ gel (1) as the .~ ~g ma~, needle injectable impl~ntc
can be readily mass pro lllre~l for a variety of hydlu~hilic or hydrophobic active
snhs~n~e with again a time in~lepPn~lent release mode.
The reciriçnt of the imrl~nt may be a hnm~n, livestûck animal inr l~ ing a rnmin~nt
~nim~l, e.g. a sheep, cow, horse, pig, goat or donkey, poultry, e.g. chicken, turkey,
goose or game bird, a la~,olatc,ly test ~nim~l, e.g a rabbit, guinea pig or mouse,
Co",~-Z "ion ~nim~l e.g. dog or ca~, or a wild animal in the captive or free state.
minictration of the imrl~nt may be by any co~vc;-lient means but is generally byinjection via the h~LldVenOIlS, i~L~d~-;lo~ l, intr~m-lcc-ll~r, sub-c~lt~n~ous or
intradermal route. The device may also be surgically imrl~ntefl or imrl~ntecl by sub-
surgical procedures such as during biopsy procedures. Devices such as these may aLo
30 be ~.l",i"i~ered by an oral route.
.
The amount of active ingredient used in a given imrl~nt will vary depending on the
~Wo 94/031S9 21~14 5 9 PCI/AU93/00392
'~ f;
_ 9 _
type of bioactive molecule, con~lition in the animal being treated and the presence
or ~hsçnre of agonists to the active ingredient or ~nt~gonicts to the con-lition being
treated. In general, an effective amount of active ingredient is employed meaning an
amount effective to in~ln~t, stim~ te, promote or otherwise initi~te the imrnediately
S intPn-ied result~
, ~
For tY~mp1e, if the active ingredient is an antigen, the effective amount is that
required to stim~ te an imm1mr rt.~l-~c to tihe ~ntig~on Commonly, the active
ingredient will be present in amounts ranging from a few micrograms to gram
10 quantities per imrl~nt
The invention will be further described by r~relellce to the following non-limiting
figures and ~ s I~ the figures:
Figure 1 is a part section~1, part srhtm~tir, cross section of an apparatus
15 suitable for ~ g imp1~nt cores by rhrmis~lly in-l11recl gelling.
Figure 2 is a part section~1, part srhtm~tir cross secti~ n of an apparatus
s1~it~hle for ~ari~g imp1~nt cores by te~ldlule in-lurecl gelling.
Figure 3 is a srhrm~tic illu~Ll~Liol. of spray co~ti~ of hydrogel impl~ntc.
Figure 4 is a schematic illustration of rod co~ting solvent based polymer
20 co~ting app1ir~tion
Figures 5 and 6 are graphs of dye release from 35% (pLa i.v. 1) to1l11ene rod
coated gelatine core imr1~ntc; in vitro at PBS at pH 7.2, 37 C.
Tn~l~nt Y~ ;
Apparatus suitable for use in the method of the ~csellt invention varies depenrling
whether the gel results from tG~lature or rh~-mic~1 effects. Figures 1 and 2 are30 part section~l~ part srhrm~tir cross sectionc of the a~ Lu~ for preparing imp1~nt
cores. In one embo-limrnt i~lustrated in Figure 1 for rhrmir~11y in-i11refl gelling, the
a~dld~us concictc of a dialy is tube "1" fitted over an upper end portion "2" and a
WO 94/03159 ~ i PCr/AU93/00392 ~
2 1 ~ 9
- 10 -
lower end portion "3" and located in sealing engagement by wate:,~rwf rubber
sealants "4" and "5". A ~u~ ing line forrneci of bioerodible core material "6" is
m~int~ine~i under ten~ion by a spring steel bow "7". An inlet port "8" and bleeder
hole "9" are provided respectively in the lower and upper end portions "2" and "3".
S An outer perforated mould "10" is provided to ~u~ l the dialysis tube "1" and allow
the ingress of geliing reagents to the dialysis tub~. ~is outer perforated mould may
be hinged to allow easy access to the dialysis tube.
In an alternative embodiment illustrated in Figure 2, s~lit~hl~ for te~ u~e
10 in~ ceci gelling, the dialysis tube "1" is repl~ced with a teflon tube "11" which may
be in two longitllriin~l halves and the outer perforated mould "10" is replaced with
a j~t~keteci te.n~e~-ature control member "12".
For structural stability of the gel during subsequent processing the gel structure is
15 held on the ~u~w ~ "6". For symmetry and ~ ~ the ~u~ "6" is centred using
the upper and lower end portions "2" and "3". The ~u~ line "6" is kept taut using
a device "7" made of spring steel r~os~mbling a bow. This device serves also as a
handle to Lla~ the gel structure for the coll~..;Pnc~e and security of subsequent
gel l~luce~ g, e.g. drying and co?tin~
Referring to Figures 1 and 2, a central su~ which forms the bioerodible core "6",
e.g. a surgical suture, is p~citiontod at the radial centre in a cylindrical mould "1" or
"11" using a~ ,yliate pocitirming guides. For water based polymeric matrices, the
inner s~ e of the mould should be lined with material such as teflon to f~ilit~te
25 cast removal.
A temperature se~ ive impl~nt hydrogel layer or core matrix on the central ~ L
may be prepared in the following m~nner:
Insert the hol;"~ lly sectioneci teflon tube "11" into the j~rk.o.tecl
temperature control member "12" with a~.. ,c;.n~t~ly 1.0 cm of the
teflon tube protruding from one end of the glass tube.
2 Run the ~u~l l line "6" through the bottom end portion "3" and knot
~ W 0 94/031~9 ~ 1 4 1 ~ 5 9 PC~r/A U93/00392
on the external side of the end portion "3" to ensure the line does not
pull through (see Step 9 below).
3 Pass the support line "6" through the mould (teflon tube "11" in the
glass holder "12").
4 Insert the protruding section of the teflon tube "11" (from step 1) into
the bottom end portion "3" (the analogy used was "like a condom").
S Push the teflonlseptum plug "11"/"3" into the glass tube "12" as far as
possible (on the whole this remains 'outside' the glass tubing).
6 Seal the teflon/septum plug "11"/"3" by çn~cing the glass tube and
septum end in the watel~oof rubber sealant "4", e.g. wrap in
parafilmTM.
At this point either of two procedures may be followed. That is, either the
mould is filled and the top end cap "2" put into position and the whole device
sealed. Alternatively, the top end cap "2" is put into position and then the
mould is filled and sealed.
7 Fill the mould with the required gelling sollltinn using a syringe.
8 Run the top end pUl Lion "2" onto the ~u~Ol ~ line and insert the top
end portion "2" into the top of the teflon/glass tubing "11"/"12".
9 Hang the entire moulding unit from the ~u~ line to align the
~u~ line "6" along the centre of the mould.
10 When cured ~licm~ntle the mould and pull out the SU1JPU1L line "6" with
the core matri~c ~tt~ e~
(2) Tmpl~nt Hy~rogel ('nre Pre~ tion
The matrix active ingredient and the hydrogel material in the gelling mix is
colll~ounded accor~ g to the individual formlll~tions used. Usually the
sollltionc of hydrogel and active materials are ~ic~ed separately and
recoml,illed in the ~ ~l Lion prescribed imm~ tely prior to filling into the
mould to minimice any possible clen~nlration or inactivation of the active
W O 94/03159 ~ PC~r/AU93/00392 ~
2 ~
- 12 -
during the preparation of the hydrogel solution.
(i) T.o."~r~tllre in-lnrr~l gellin~g pro(rcc
S The assembled mould, the gelling mix and the filling device (eg a syringe) are
separately equilibrated to the desired fi~Lng temp L d~l~e. Thermal prefilling
equilibration is desirable to prevent bI~kage during filling and deformities
in the gel structure.
2a The plc~alation of the tem~.a~ in~hlreci gelling solution involves
diccolntion of a hydrogel, such as gelatine (270 mg), and, if required,
an osmotic modifier such as sucrose (750 mg) in water (1.5 ml). The
stock hydrogel solution is ~ aled by he~ting the suspended solutes
in a water bath at 100C. Subsequent to rlicsollltion, fluidity of the
hydrogel sollltion is best m ~int~in~ll by hokling at 37 - 45 C.
2b A stock 'bioactive' sollltion is ~ cd by flic~ol~ltinn of the 'active' in
water (0.5 ml). This s~ ltinn is then held on ice.
2c ~ nrc~cc~ry, the requisite volume of the stock 'bioactive' solution is
diluted to the lc~ e cQ..- e ~ iull by ~icsollltion with water. This
snllltion is also held on ice.
2d 0.5 ml of the stock 'bioactive' solution or diluted 'bioactive' solution isheated to 37C then combined with the stock hydrogel solution (1.5
ml) to give the gel sohltinn The combined hydrogel and active agent
solutions are then mixed for 1 minute at 37 C and dispensed into the
moulds.
2e The gelling mi~ may be introduced to the mould by a syringe pump via
the inlet "8" while the bleeding hole "9" provides an outflow for air and
excess gel solution. Filling is ~refeldbly ~ccomrli~hed by one slow
collt;.l..ous action. Byfi~lingfrom bottom ~w~Ld the process is made
e~çnti~lly trouble free and there is lesser likrlihood of deformities in
the gel structure due to ocrlllded air bubbles especially in a clean
teflon mould. Filling is ~mplrted when gelling mi~c appears to flow
~O 94/03159 21 ~1 4 5 9 Pcr/Au93/oo392
. .
- 13 -
from the outlet bleeding hole in the top positioning guide.
2f Foliowing filling the comr1ete ~cs~omhly is m~int~ined at the filling
t~ ature for a short time and a check for proper filling is made.
2g Gelling of the matrix/active solution mix is ~ccQmr1iched by lowering
S the ambient tem~l~ture in the thermal jacket slowly. A slow
transition is desirable to ~ ellt defc.lmiLies in the gel structure. To
ensure good results, the u~mr1ete ~cs~ombly is kept at a temperature
well below the critical gelling temperature for a period of time, for
~mrl~ 30 ~ t~ s.
(ii) (~h~mir~lly or ion in-ln~.o~l ~11in~
For polymeric matrix materiaLc which forrn a solid gel when in contact
with a di- or trivalent cation or a catalytic agent, the supporting string,
is centred by the ~iLiOllillg guides "2" and "3". The Su~l l line "6" is
kept taut using a device "7" within an a~ro~liate dialysis tubing "1"
which in turn is çn~ se~i within a rigid ~Çolatel ~ ~ll "10" for the
e--;enl e of filling and ~ l)se~luent in~ nn of gelling.
20 (3) Filling
3a Ion or ~h~.~ni~lly in~ e~i gelling is best achieved ucing the perforated
mould ass~ ly "10" with filled dialysis tube "1". This assembly is filled
~ler~lably by one slow co,~l;"llous action. By filling from bottom
u~w~d the ~rocess is made .o.ccenti~lly trouble free and there is lesser
lik~.lihoo-l of derulllli~ies in the gel structure due to ocç111rled air
bubbles a&ering to the dialycis tubing.
3b Filling is c~mp1eted when gelling mi~ appearc to flow from the outlet
bleeding hole in the top positioning guide.
3c Following filling the complete ~c.c-o.mhly is m~int~ine~i at the filling
tem~ ature for a short time and a check for proper filling is made.
3d The assembly is then ~r~ed to a c~t~er of the in~l11cer solution
WO94/03159 ~,~4~4S9 PCr/AU93/00392 ~
- 14 -
~vhich may be held at any ~,erelled temperature, for example, at zero
degrees Celsius if den~lration of an active is a concern. Equilibration
of the in~lllrPr across the semi-permeable membrane affords the
gelling of the matrix active solution mix.
3e Extra time should be allowe~ to ensure proper gel formation and
possible hardening of the ~ structure for ~Y~mplP, 240 mimlt~os"
(4) Remov~l of gel sh~l-hlre from monld
Depending on the gel structure strength, fresh gel structures on the ~u~po
are usually very fragile and thus not readily lifted ho~ .t~lly off the mould.
Hence they are ~,~f~lably kept in a vertical position and the t~vo halves of
the mould gently se~alated. To ~ n~ m~ging the gel structure, the top
po~itioning guide should be removed before the removal of the mould halves
but the bottom guide should remain in place.
(S) ~ gment~tion of the ~el ~h~l~hlre
The moulded gel structure lerc.l~d to above c4..~ c of an outer hydrogel
layer which is ~up~l~ed by and ~-~ .I.ic about an axial ~u~oll line.
.~m~ller discrete gel se~ P~t~ which remain ~u~lLed by the axial ~u~
line can be created by cutting the outer ccs--~..l~ic hydrogel layer in such a
way that the axial line remains intact and sliding the cut segrnent along the
axial line so as to form a space bet~veen the disc,~ete small gel segments. The
above cut/slide operation may be ~lrollL~ed either before or after the
concentric hydrogel layer has been dried and prior to Co~ting Huw~ver, it
is generally easier to perform the cut/slide operation on wet hydrogel.
(i) The "long section" may be segm.onted into lengths, e.g. 10 mm,
m~int~inP~ on the support line "6". The ~U~Ul led segments may then
be dried and processed as required for specific applic~tions.
~WO 94/03159 21414 5 9 i pcr/Aus3/oo392
- 15 -
(ii) In an alternate emborliment, the core matri~ may be sectioned
subsequent to drying but prior tO coating.
(6) Drying
5
Depending on the molec~ r stability of the active cu~ oulld and speed of
drying, the water based gel structure may be air dried under ambient
con~itir)nc or at low te~ atures (e.g. at 2 C) by flllching with dry nitrogen
gas. For ~ mrle, a gel structure CQ~ g about 10~o total dry matter takes
8-10 hours to dry the gel to a co~la,lt weight at room temperature (21 C).
The drying time will be appreciably longer at lower te~l alul es. The drying
times are gel co~ o~ ion Clepen~ nt
(7) (~o~tin~ of ~he (~Jt~ tllre
(i) Once dried the gel structure mounted on the support may be
re~cct~mhled with the 2 halves of the teflon mould of the same internal
or dirrelc.,t internal (li~nnett~r to allow for the bioerodible outer
co~ting to be ~rrliecl to the core. Thus, after the ca~ct is formed and
dried, the dry cast may be coated with a water ;.,.~.~ .".eable co-
polymer such as polylactic acid (pLa)/polyglycolic acid (p&a) (85:15)
co-polymer to form a co~ting
(ii) Alternatively the dried gel structure on the su~l l "6" may be coated
by repeated dipping into a polymeric sohltion and drying to achieve a
thin layer of a s~ "l i~lly water i~ ~eable coating with specific
tl~S~ characteristics (refer to Fy~mple 3).
(iii) Alternatively the dried gel structure on the ~u~ "6" may be coated
by repe~tecl ~Lyillg with a polymeric solution and drying to achieve
a thin layer of a s~ st~nti~llywater impf-rme~ble coatingwith specific
characteristics (refer to F~mrle 4).
Wo94/03159 ~,~4~4S9 PCr/AU93/00392 ~
- 16 -
(iv) Alternatively the dried gel structure on the support "6" may be coatedby repeated applir~tion of a polymeric solution via a "rod" or '~vick"
and drying to achieve a thin layer of a snhst~nti~llywater impermeable
coating with specific 11 ~a~ characteristics (refer to F~mple S).
(8) ~ ce Profile Control
For manip ll~ting the release characteristics, the release mode of the present
device can also be modified by the cQn~e~ alion and com~osition of the
matrix materials. For ~y~mrle~ when agar was used as the matrix material, the
release was faster and the extent of release higher. This demonstrates the
ability to affect the release rate by changes in matri~c composition. Variationsin~ lcle repl~çment of the water based hydrogel matrix with hydrophobic
materials such as glycerol monoste~rate.
lS
l;XAlVl P~ .F. 1 - Fabrication of imrl~nt cu~ human serum albumin (HSA) andhnm~n IgG
This ~Y~mrle is illL~LraLi~.. of te~lature in~hl~ecl gelling and gives evidence of an
20 i ~ ogical les~ollse to an incc,l~l ated ~ntigen
Flc;y~,~t;nn of Tmpl~nt
0.9 ml of HSA/hllm~n IgG soll~tion (9.5 and 5.8 mg/ml respectively in 0.1 M NaCl)
2S were miYed with O.S ml of 1~o NaCl solution and the mixture equilibrated to 43 C.
It was then mixed with 4.6 ml of gelatine/agar matri~c solution (13% and 1.3% w/v
respectivel~, gel~tine, cell culture reagent from Porcine stein, approx 300 bloom,
Si~na catalogue no. G1890) also equilibrated at 43C. The reclllt~nt mixture wasd to the mould assembly eguilibrated at 43C with a 7 mm internal
30 diameter x 20 mm long teflon mould and a 0.4S mm cli~meter ~" line as the
centre ~uy~o- l. The inlets to the moulds were then stoppered and the moulds were
allowed to e~uilibrate at 43C for 10-1S minlltes before the temperature of the
~O 94/03159 2 ~ 4 1 ~ 5 9 PCI /AU93/00392
,
- 17-
thermal jacket was slowly lowered to that of mnning cold tap water at about 20 C
over 20 mimlteS. The gel was then soliclified by cooling the mould assembly to 0 oc
and m~il.t~ i..g it at that temperature for half an hour. After the mixture soli~lifiecl
the moulds were r1icm~ntled and the gel structure on a string stretched in a bow was
S removed carefully in a vertical orient~tion to avoid hc,l~ullLal splitting of the gel
structure by the stretched ~u~ while the gel structure was still fragile. The gel
structure ~UypOI led by the string ~up~ll in a bow were cut and separated in thecut/slide operation and air dried at room temperature overnight (about 18 hours).
The dried gel structures were then coated with a water based polyacrylic resin
10 Eudragit E 30D by five repeated cycles of dipping in an Eudragit suspension for 30
seconds and drying for 60 minlltes~ Eudragit E 30D is an aqueous dispersion of
poly(meth)acrylic acid esters supplied by Rohm Pharma GmbH Weiterstadt
Darmct~lt West Germany. When dry, the gel structures were dismounted from the
bow and trimmed to se~m~-ntc of 1 cm length (diameter 3 mm). Each segrnent was
15 estim~terl to co.-l~;.. 0.051 of the original HSA/h-lm~n IgG antigen solution.
Placebo imrl~ntc were made in exactiy the same way as the antigen impl~ntc except
that the HSA/hl~-n~n IgG sol-~tinn was replaced by 0.9 ml of 0.1 M NaCl.
20 Fv~ tinn nf Tmrl~nt
Sheep previously ;~ cd with human serum were imrl~ntecl subcutaneously on
the inside sulr~ce of hin-lle~c using a me~h~ni~l imrl~nt~r fitted with a 2.8 mln
internal ~ met~r n~eclle- Two im~l~nt segm~ntc co..~ -g antigen or placebo were
25 given to each sheep.
To determine the antibody les~,.ce) blood s~mpl~s were taken from the sheep prior
to receiving imrl~ntc and on days 9, 15 and 23 following impl~nt~tion. Sera wereobtained from blood s~mrles by standard sernlogit~l practice and assayed for
30 ql-~lit~tive and qll~ntit~tive antibody titres using double immllno diffusion and
ELISA (Enzyme T.ink~l Tmmllno Sorbent Assay) te~hni~lues respectively.
2 ~ 4 '~ ~ PCr/AU93/00392
- 18 -
Rt~cnl~c
Results of double immnno diffusion assay showed that there was an increase of
antibody titre in recipients of the HSA/human IgG imrl~ntc but not in those of the
5 placebo. The increase in antibody titre was r~ .able to that obtained when
norrnal immnnic~tion protocol with adjuvanted antigen ~us~nsion was used. As
shown in Table 1, the le;,~ollse in general wac m~rim~l at day 9 of the four
scheduled s~mrling days for both the specific anti-IgG and anti-HSA responses.
~ im~l increases at day 9 ranged from four to thirteen fold for anti-IgG re.,~l~sc
10 and seven to twent~five fold for anti-HSA. Titres were notably high relative to
those in sheep receiving adjuvanted antigen suspensions, with amounts of specific
antibodies approaching the highest that has yet been obtained from a few select
sheep (that is in the order of 32 units of antibody in the sheep serum to either the
HSA or human IgG). For three weeks following imrl~nt~tion, antibody titres
15 rem~inecl signifil ~ntlyhigh but decreased with time in the antigen imrl~nte~ ~nim~lc
In addition to the at least equivalent 1 ~s~ es obta~cd, i l l l l l l ll i~ i.c~tion using antigen
imrl~ntc can afford a ~lu~ber of other alY~tages. T...~ i.c~fion with imrl~nt.c was
very simple to ~....1~,1l~ and cr....~.~lcd with the louLiL.c method far less time
20 co.. ~.. ing Apart from the puncture mark caused by the imrl~nt~r needle, no
ulceration or swellingwas evident at the imrl~nt site. This is in sharp contract to the
sitll~tion of rouli~e practices when ~nti~n ~u~ innc, especially those using
Freund's adjuv~, are used for ;.~ ...ic~ti.m
~0 94/03l59 2 ~ 59 Pcr/Au93/oo392
- 19 -
TABLE 1 - Qll~ntit~tive Antibody Response by ELISA Method of Deterrr~ination
Sheep Tmrl~nt Day O Day 9 Day 15 Day 23
No.
S Anti-IgG Re~ .se
7 Antigen 3.3 19.9 (6.0X) 15.2 (4.6X) 9.6 (2.9 X)
9 Antigen 1.7 1?.8 (10.SX) 14.0 (8.2X) 9.9 (5.8X)
3 Antigen 0.9 11.7 (13.0X) 8.9 (9.9X) 6.4 (7.1X)
O Antigen 7.0 24.9 (3.6X) 28.3 (4.0X) 25.3 (3.6X)
8 Antigen 1.2 8,5 (7.1X) 6.9 (5.8X) 5.0 (4.2X)
Placebo 2.0 2.0 (1.OX) 2.2 (1.lX) 2.1 (1.lX)
6 Placebo 4.8 8.8 (1.8X) 8.2 (1.7X) 6.1 (1.3X)
4 Placebo 3.9 4.1 (1.lX) nd- nd-
Anti-HSA R~ se
7 Antigen 1.6 15.7 (9.8X) 13.2 (8.3X) 8.0 (5.0X)
9 Antigen 1.0 21.1X (21.1X) 19.4 (19.4X) 14.5 (14.5X)
3 Antigen 0.9 22.3 (24.8X) 20.6 (22.9X) 14.2 (15.8X)
O Antigen 3.5 23.8 (6.8X) 26.6 (7.6X) 18.9 (5.4X)
8 Antigen 1.5 13.3 (8.9X) 12.2 (8.1X) 9.8 (6.5X)
Placebo 1.0 0.9 (0.9X) 0.9 (0.9X) 0.9 (0.9X)
6 Placebo 4.9 6.4 (1-3X) 6.0 (1.2X) 5.8 (1-2X)
4 Placebo 1.7 1.5 (0.9X) nd- nd-
nd = not det~ ~ Illilled; figures in brackets l~lesent the mllltirle increases over day O
WO 94/0315~ ~ 4~ 4S 9 PCr/AU93/00392
- 20 -
.F. 2
This ~ m~le is illustrative of chemical in~lllre~ gelling and is preferred for heat
labile active material.
The ion in~ cerl gelling mould was assem~ed as described above (refer to Figure 1)
and equilibrated at room tempelaLure. 7 ml of 3% w/w aqueous sodium ~lgin~te
[Sigma Co. cat No. A-2033, alginic acid sodium salt medium viscosity, from
M~rrocystic ~yl ;r~ (Kelp)] was mixed thoroughly with 7 ml of (~loctrit1illm nnvyi
10 toxoid at room te~ ature. The active ~uspel~sion was a concentrated solution of
~lnctrirlillm ~ toxoid cc~ llg the toxoid produced by the bacteria and 5.3
mg/ml of hydrated ~lllminillm hy~l~ide added as adjuvant. Although there is a
slight increase in viscosi~y, the two ~ ion~ may be held at lower temp~ es
(eg 4C) and mixed at lower telll~ldlL~es. The reslllt~nt mixture was transferred
15 with a syringe via the inlet port in the mould ~cc~mhly to fill the dialysis tube inside
to a slight overflow from the ouclet bleeder hole with about 7 ml per mould. The two
mould ~c.c~mhlies were ~i~.~ed into a bath of 500 ml aqueous AlCl3 sollltinn
(0.5% w/w) at room tempe~a~ul. (or if desired at 1C) with continllQus slow
~git~tion using a m~gn~tic stirrer. Gelling was usually effected within 2-3 hours of
20 imm~orsinn but it is our nnrm~l practice to leave the gel structures to form for 4
hours. Following gel structure form~tion the moulds were removed from the gels in
a vertical position The reslllt~nt gel structures were allowed to dry at room
te~ dlule. When dry, the gels may be surface coated with Eudragit E 30D as
previously described and/or reloaded with another layer of toxin ~Igin~te gel
2~ structure.
If desired sodium ~l~in~te may be directly ~lb~l;t~n~d by K-carrageenan but the
tempelalule during filling of the tube mould needs to be higher to l~leV~llt
premature gelling of the K-carrageenan.
Also if rapid gelling of the matrix active sollltinn is required, the gel inr~ ing agent,
AlCl3, (e.g. 7 ml of 1.5% snllltinn) can be added directly to the 14 ml of the ~Igin~te
~Wo 94/03159 ~ 4 f 9 5 9 pcr/Au93/oo392
~. n~vyi mi~ and the rtos..lt~nt solution mixed thoroughly and quickly.
l~XAlUP~ F 3
S This loY~rnrle illustrates the terhnique of dip coating of hydrogel irnplants and
provides evidence of pulsatile release in vitro ev~ tion
Polylactic acid (d,l-pLa) of inherent vi ,oosi~y (i.V.) = 1.0 dl/g (supplied by
Boehringer Ingelheim) of mass 12 g wac dissolved in 120 ml of dichlorometh~n.o
10 Phosrh~te l~u~Lred saline (PBS), pH 7.2 was made accoldillg to the following recipe:
NaCl (80.00 g), KCl (2.00 g), 2HPO4.12H2O (15.36 g), KH2PO4 (2.00 g)
dissolved in 1000 ml of lictilleri water.
The l~ ogd cores cQnt~ining red food dye or antigen were m~nllf~rtllred according
15 to the methodology described above using the cut/slide method.
Refel~g to Figure 2, dried hydrogel cores on the ~u~ line "6" attached to the
spring steel bow "7" were dipped into the sollltirm of polylactic acid in
dichloromçth~ne. Tmmersion time was 30 secon-ls for each coat. Upon removal from20 the polymer s~ ltion the excess polymer sollltir)n was allowed to run down the
imrl~ntc and the hanger was then inverted to allow the polymer to run the other
way: this aids in spreading the polymer evenly over the impl~ntc As illustrated in
Table 2, groups were dipped either four times, i.e. 4 coats or si~c times, i.e. 6 coats.
At least one hour drying time was allowed bet~veen coats to ensure adequate drying
25 under the ~mbient conr~ on~. Individual cut impl~ntc were each placed in individual
glass vials. PBS of volu_e 10 ml was added to each vial and they were placed in an
incubator at 37 C. Molli~o~g wac carried out using U.V. visible spectroscopy tOme~cllre dye release into the solution.
S pcr/Au93/oo392
- 22 -
TABLE 2
itions
Number ofNumber of Coats T~ erature (C) pH
Sarnples
7 4 37 7.2
7 6 37 7.2
10 One sample commenced dye release early at day 4 and took 14 days tO attain 100%
release at day 18.
Of the rem~ining replicates mean release was at day48. The same impl~ntc attained
a 100% release profile in an average of 5.3 days +/- 2.7 days. All s~mples
15 commPn~ecl dye release within a si~c day period bet~veen days 45 and 51. The total
release time from when the first of the six impl~nt~ comm~n~ed dye release ( ~10%
release) to when the final imrl~nt reached 100% release was 14 days, i.e. from day
45 to day 59.
20 There appeared to be no st~tictic~lly signifir~nt ~lirrGlGnce between s~mrl~c with 4
coats and those with 6 coats. This is ~ ected as the pLa polymers are, in general,
buL~c eroding thus are generally unaffected by sarnple thickn~ss
The results show a ~ignifi~ ~nt delay in the time of onset of release of die comr~red
25 to uncoated s~mrl~s which gave 100% release within 3 hours of imrnersion in PBS.
F XA~IPF F. 4
- This ~r~mrle illustrates spray coating of hydrogel impl~nts and provides evidence of
30 pulsatile release from an in vitro eY~min~tion
~WO 94/03159 21414 S 9 PCr/AU93/00392
- 23 -
Polylactic acid (d,l-pLa) of inherent viscosity (i.v.) = 1.0 dl/g (supplied by
Boehringer Ingelheim) of mass 12 g was dissolved in 120 ml of dichloromethane.
Phosphate buffered saline (PBS), pH 7.2 was made according to the following
recipe:
NaCl (80.00 g), KCl (2.00 g), 2HPO4.12H2O (15.36 g), KH2PO4 (2.00 g)
dissolved in 1000 ~nl of distilled water.
The hydrogel cores cont~ining red food dye or antigen were manufactured according
to the methodology described above using the cut/slide method.
An ~tomicing spray nozzle connt~cted to a ~""u,essed air cylinder and to a 250 ml
separating funnel which cont~ine~l the diLrerent polymer solutions was used to apply
the polymer to the gelatine cores being rotated about the ~iU~ line using a
"rotisserie" mech~nicm (refer to Figure 3). A polymer sol-ltion was sprayed onto the
imrl~nt cores using air brush with a g~c ~lcs~ of 200 kPa and a ~lict~nce of 10-30
mm bclwcen the air brush outlet to the imrl~nt cores. The imrl~nt core string was
hand rotated during Spl~yil~g in order to obtain .."ir~... co~ting. 65 ml of polymer
solution was sprayed on a string of imrl~nt cores.
20 Referring to Figure 2, the position on the ~up~l line "6" of all imrl~ntc was noted,
seven of those were selected at 1~dO1LI and their weights recorded. Tmrl~ntc ~vere
placed in glass vials. PBS of volume 10 ml was added to each vial and they were
placed in an ill~ubatol at 37 C and pH = 7.2. The d,l-PLA (iv = 0.1 dl/g) produced
an opague papery coating which had a granular appearance. Despite their
25 appearance swelling without releace was ~ale"t from an early stage.
Results of the incubation work are given in Table 3. Seven imrl~nt.c were placed in
individual s~mrle vials ront~ining 10 ml rhnsph~te buffer pH = 7.2 and incubatedat 37 C. MO~iLO1 illg was carried out using U.V. visible spc~ L~ osco~y to measure dye
30 release into the snlllti-~n One imrl~nt started to release on day 36, others started to
release between days 73 and 79.
WO 94/03159 ~,~ 4~.~S9 Pcr/A~I93/00392
-- 24 --
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~ 94/031ss ~ 1 4 1 l 5 g PCr!AU93/00392
F XA~P~ F. S
This ~mple illustrates "rod coated" solvent based polymer app1ic~tion and provides
evidence of pulsatile release in in vitro ev~ ,tion
A method of coating gelatine cores was inv~stig~tecl to try to uve~rconle the difficulty
of Cu~ lg the ends and edges of the core when spray co~ting A saturated -viscous5nl~1tion of polylactic acid (d,l-pLa) of inherent viscosi~y (i.v.) = 1.0 dl/g in
dichloromethane (20% w/v) was made. Fu~ ore a co~ e;lecl series of gelatine
10 cores ~u~,uolled on an axial ~U~1~Ul~ was made using the cut/slide operation as
described above. It was found that if one large viscuus drop of material was spread
along a slowly rotating gelatine core (a~ tely 120 rpm), the drop rPm~ine~levenly dispersed and seemed to sllcce-ccfully ~ulloL~d the sharp edges and the end
of the core (Fig. 4A). App1ic~ti-~n of the solvent based polymer was with a glass rod.
15 The rod is dipped into a viscous polymer so11ltion to pick up a small amount of
material (Fig. 4B). The mateAal is then applied to one end of the gelatine core and
drawn to the other (Fig. 4C).
Forty of these s~mrles were placed in a PBS, pH = 7.2, twenty at 37 C and twenty
20 at 50C. Results of the 50C appear in Table 4 and in Figures S and 6. Monitoring
was carAed out using U.V. visible s~ osco~ to me~cllre dye release into the
sollltion The 37C s~mrles had been in vitro for six~r dayc, the majority of which
show no signs of release (Table 4).
W O 94/03159 2~4~59 26 - PC~r/A U93/00392 *
-
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~WO 94/03159 ~2 1 4 1 ~ 5 9 PCI /AU93/00392
- 27 -
T;XAl~ F. 6
- This ~Y~mp1e shows the use of impl~ntc to obtain pulsatile release in ~nim~lc
S An experiment was performed in which the delayed release from impl~ntc of tetanus
toxoid was demonstrated by rletecting by ELISA antibodies produced by the mice in
cs~ se to the released antigen.
Tmpl~ntc were made as described in Fs~m~le 4 except that 0.5 mg tetanus toxoid per
10 impl~nt was used as the active ingredient.
Antigens were used in impl~ntc with two coat cc,l~ ;tion.c and the control group (3)
lcceived antigen in nnCo~te~ gelatine cores.
15 Mice were imrl~nteri sul~ t~.~eously on the inside sllrf~re of hind legs u~sing a
mech~nic~l impl~nter fitted with a 2.8 mm internal diameter neerile~ One impl~ntse~m-ont co~t~i..i..g ~nti~n or placebo was given to each mouse.
To dete. ., . i . .ç the a l~ly r~ c, blood s~rnrles were taken intra-ocularly from
20 the mice prior to receiving impl~ntc and on days 14, 21, 36, 49, and 63 following
impi~nt~tir)n Sera were ~ ed from blood s~rnrl.os by standard serological
practice and assayed for qualitative and ql~ l;vt; antibody titres using double
;"""."~-- diffusion and ELISA (Ellzyme Linked Tmmllno Sorbent Assay~ techniques
respectively.
FT T~S~ met~t~ri
Nunc Maxisorp microtitre plates were coated overnight at 4C with the antigen ofchoice. Test serum was serially diluted on the plate to final dilutions in the range 800
30 to 25,600, and incubated at room te~l a~ , for one hour. Plates were washed and
rabbit anlis.,. ~u to mouse i ., . ~ globulin~ conjugated with horse radish peroxidase,
was added and incubated at room te.,~ alu~`e for one hour. Pero~ e activity was
WO 94/03159 2~ ~145 9 PCr/AU93/00392 ~
- 28 -
~etectecl using 2,2'-a7ino-bis(s-ethylben7thi~7nline-6-sulphonic acid) (ABTS)
substrate.
Evidence of seroconversion and thus of thè ~elease of antigen was any positive titre
5 (800 to 25,600) obtained.
The results given in Table S in~ir~te that the majority of ~nim~lc given uncoated
s~mples showed serocollvclaion having been achieved within 14 days whereas nQ
seroco~ aion was observed with coated impl~ntc until 21 days. With imrl~ntc
10 coated with pLa homopolymer (i.v. = 1.0) se~oco~ .aion in all cases had not been
achieved even after 63 days. That 100% serocc)~ .ion will result in all cases isinrlir,~te~l from the ol>s~,lvcd eventual scl~oco~ aion of all ~nim~lc treated with
imrl ~ntc coated with 50:50 pLa/pGa (i.v. = 1.0). ~nim~lc receiving placebo imrl ~nt~
gave no antibody tire to Tetanus to~id.
TABLE S
Group No. of Coat cc~ ;on Serocollv~laion number (days)
Mice 14 21 36 49 63
1 6 d~-pLa(i.v. = 1.0) 0/6 0/6 316 316 316
2 6 d~-pLa/pGa 0/S 2/5 5/5 5/5 5/5
(50:50; i.v. = 1.0)
3 4 no coat 2/3 213 3/3 ~13 313
3 5 placebo - no c,oat 0/S 0/S 0/S 0/S 0/5
Since mo-lifir~tionc withill the spirit and scope of the invention may be readily
effected by ~l~Ol~S sldlled in the art, it ia to be understood that the invention is not
limite(l to the particular embo-limrnt des~;libcdl by way of .oY~mplç, hereinabove.
~0 94/031S9 ~ 1 4 1 ~ 5 9 PCr/AU93/00392
- 29 -
REF~RENOES:
1. R. Jeyanthi and K. Pandurange Rao, "Controlled Release of Anti-Cancer
Drugs from Collagen-Poly (HEMA) Hydrogel Matrices", Journal of
S Controlled Release 13 (1990), 91-98.
2. Y.F. Maa and J. Heller, "Controlled Release of 5-Fluorouracil from Linear
Poly (Orthoesters)", Journal of Controlled ~2ele~e, 13 (1990), 11-19.
