U.S. patent application number 11/148662 was filed with the patent office on 2006-01-19 for injectable microspheres.
Invention is credited to Joseph Carozza, Chih-Chang Chu, Daqing Wu.
Application Number | 20060013886 11/148662 |
Document ID | / |
Family ID | 35599723 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060013886 |
Kind Code |
A1 |
Wu; Daqing ; et al. |
January 19, 2006 |
Injectable microspheres
Abstract
Injectable microspheres are obtained from double bond
functionalized polyhydric alcohol ester by a method comprising
dissolving the double bond functionalized esters in a hydrophobic
organic solvent, forming an aqueous solution of stabilizer, forming
an oil in water emulsion where the solution of stabilizer
constitutes the continuous phase and the solution of ester
constitutes the disperse phase and evaporating the organic solvent
or from block copolymer of PGCLM and methoxy poly(ethylene
glycol).
Inventors: |
Wu; Daqing; (Ithaca, NY)
; Chu; Chih-Chang; (Ithaca, NY) ; Carozza;
Joseph; (Southport, CT) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
35599723 |
Appl. No.: |
11/148662 |
Filed: |
June 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60582824 |
Jun 28, 2004 |
|
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Current U.S.
Class: |
424/489 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 9/1647 20130101; Y10T 428/2982 20150115 |
Class at
Publication: |
424/489 |
International
Class: |
A61K 9/14 20060101
A61K009/14 |
Claims
1. A biodegradable microsphere formed from multiarm biodegradable
polymers wherein the surface of the microsphere is hydrophilic and
comprises unsaturated C.dbd.C bonds, wherein said microsphere is
formed from a double bond functionalized polyhydric alcohol ester
of polyester, and wherein said microsphere can be loaded with at
least one bioactive agent.
2. The microsphere, according to claim 1, wherein the surface of
the microsphere is converted to a hydrogel.
3. The microsphere, according to claim 2, comprising a bioactive
agent encapsulated in the microsphere, as well as a bioactive agent
entrapped within said hydrogel.
4. A method for preparing a biodegradable microsphere wherein said
method comprises (a) dissolving a double bond functionalized
polyhydric alcohol ester of polyester in a hydrophobic organic
solvent, (b) dissolving a stabilizer in water, (c) admixing the
solutions formed in step (a) and step (b) to form an emulsion where
the solution formed in step (b) constitutes the continuous phase
and the solution formed in step (a) constitutes the disperse phase,
(d) evaporating the organic solvent to form a hardened microsphere
by polymer precipitation, from the double bond functionalized
polyhydric alcohol ester, (e) recovering the microsphere.
5. The method, according to claim 2, wherein the stabilizer is a
polyalcohol.
6. The method, according to claim 4, wherein the stabilizer is
dextran.
7. The method, according to claim 4, wherein the stabilizer is a
sucrose ester.
8. The method of claim 4 where the double bond functionalized
polyhydric alcohol ester is obtained by polymerizing
.epsilon.-caprolactone monomer or a blend of .epsilon.-caprolactone
monomer and lactide monomer or glycolide monomer in the presence of
polyhydric alcohol containing from 3 to 6 hydroxyl groups to form
polyhydric alcohol ester where the acyl groups contain free
hydroxyl at their terminal ends and reacting with maleic anhydride
to convert some or each of the free hydroxyls to moiety containing
2-carboxy ethenyl group.
9. The method of claim 4 where the double bond functionalized
polyhydric alcohol ester is obtained by polymerizing
.epsilon.-caprolactone monomer in the presence of glycerol to form
the polyhydric alcohol ester where the acyl groups contain free
hydroxyl at their terminal ends and reacting with maleic anhydride
to convert some or each of the free hydroxyls to moiety containing
2-carboxy ethenyl group.
10. The method of claim 4 where the double bond functionalized
polyhydric alcohol ester has a number average molecular weight,
M.sub.n, ranging from 1,000 to 50,000.
11. The method of claim 4 where the solvent is one that dissolves
the double bond functionalized polyhydric alcohol ester at room
temperature and has a boiling point ranging from 30-45.degree.
C.
12. The method of claim 11 where the solvent is
dichloromethane.
13. The method of claim 4 where the volume ratio of solution formed
in step (b) to solution formed in step (a) admixed in step (c)
ranges from 3:1 to 10:1.
14. The method of claim 4 where a photoinitiator is included in the
solution formed in step (a) and the admixture formed in step (c) is
irradiated to obtain photocrosslinking to provide hydrogel surface
on disperse phase particles.
15. The method of claim 4 where water soluble drug or other
biologically active agent is dissolved in water to form an aqueous
solution which is admixed with solution formed in step (a) to form
a water in oil emulsion which is admixed with the solution formed
in step (b) to form a water in oil in water emulsion in step
(c).
16. The method of claim 4 where oil soluble drug or other
biologically active agent is present in the solution formed in step
(a).
17. The method, according to claim 4, wherein a double bond
functionalized poly (ortho ester) is prepared by the reaction
between the diketene acetal
3,9-diethylidene-2,4,8,10tetraoxaspiro[5.5]undecane and
1,10-decanediol or triethylene glycol.
18. An injectable microsphere having a mean transverse dimension
from 5 to 200 .mu.m, formed of a hardened double bond
functionalized polyhydric alcohol ester of polyester loaded with
from 0.1 to 25% by weight of the microsphere of a drug or other
biologically active agent for sustained release after injection of
the microsphere where the surface of the micropshere has micropores
formed therein.
19. The injectable microsphere of claim 18, wherein the double bond
functionalized polyhydric alcohol ester of polyester is obtained by
polymerizing .epsilon.-caprolactone monomer in the presence of
glycerol to form polyhydric alcohol ester where the acyl groups
contain free hydroxyl at their terminal ends and reacting with
maleic anhydride to convert some or each of the free hydroxyls to
moiety containing a 2-carboxy ethenyl group.
20. A drug-loaded microsphere formed from an amphiphilic plural
block copolymer where one block is from PGCLM and the other
block(s) are from methoxy poly(ethylene glycol), by dissolving the
copolymer in organic solvent, adding the drug to the formed
solution, then removing free drug and solvent and then drying.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority to U.S. Provisional
Application Ser. No. 60/582,824, filed Jun. 28, 2004, which is
hereby incorporated by reference herein in its entirety, including,
any figures, tables, or drawings.
TECHNICAL FIELD
[0002] This invention is directed at materials and methods for
forming injectable hydrogel microspheres and at the microspheres
formed there from that can be used to load drugs and other
biologically active agents and are useful for controlled release of
these in the body.
BACKGROUND OF THE INVENTION
[0003] Microspheres with encapsulated or covalently bonded
biologically-active agent allow provision of an injectable
suspension as a substitute for surgical implantation and facilitate
administration of multiple drugs in a single injection. These
microspheres provide an initial burst to reach a therapeutic
concentration followed by a zero-order release of drug to maintain
the therapeutic level by compensating for metabolic loss. The
microspheres thus provide a sustained release therapeutic
concentration.
[0004] Microspheres of biodegradable polyesters from
D,L-lactide/glycolide and microspheres of biodegradable polyesters
from .epsilon.-caprolactone have received attention for controlling
release in the body of pharmaceutical agents and macromolecules.
However, these polyesters are relatively hydrophobic and a more
hydrophilic surface is desirable on an injectable microsphere to
increase effective lifetime in the circulatory system and to reduce
the occurrence of an inflammatory response. Hydrophilic
characteristics have been achieved by surface modification of the
polyester microspheres with hydrophilic polymers. Polyester
microspheres with more hydrophilic surfaces have not heretofore
been obtained without relying on chemical attachment or physical
absorption of hydrophilic polymers.
BRIEF SUMMARY OF THE INVENTION
[0005] The subject invention provides unique and advantageous
biodegradable injectable polyester microspheres. The microspheres
of the subject invention can be prepared with surface
hydrophilicity, but without the requirement of surface modification
with a hydrophilic polymer. In a preferred embodiment the
microspheres are formed from a double bond functionalized
polyhydric alcohol ester of polyester.
[0006] In one embodiment, the subject invention provides
biodegradable microspheres from multiarm synthetic biodegradable
polymers. Specifically, a novel drug carrier composed of
double-bond-functionalized glycerol poly(.epsilon.-caprolactone)
maleic acid (PGCLM) microspheres can be prepared by an
emulsification-solvent evaporation technique. The microspheres have
unsaturated C.dbd.C double bonds on the surface. The availability
of this chemical functionality on the microsphere surface provides
enormous potential for altering the surface chemistry of the
microspheres to tailor to specific clinical applications. For
example, the C.dbd.C double bonds can be used to render a hydrogel
network surface on these microspheres via a photo-crosslinking
treatment. The resulting microspheres then have two very different
compartments: a hydrogel surface with a non-hydrogel core.
Different drugs can be incorporated into different compartments for
customized target release.
[0007] In a specific embodiment, an ovalbumin (OA) protein was
pre-loaded into the synthetic biodegradable microspheres of the
subject invention and its release was examined. The microspheres
were characterized in terms of their morphology, size distribution,
drug loading efficiency and stability. Results shown that OVA was
successfully entrapped inside the microspheres with loading
efficiency up to 45% (w/w) and loading level 8.1%. The cumulative
OVA release % (w/w) reached around 40% at 37.degree. C. in 50
days.
[0008] In one embodiment, the subject invention is directed to
forming a biodegradable injectable microsphere by a method
comprising the steps of:
[0009] (a) dissolving a double bond functionalized polyhydric
alcohol ester of polyester in a hydrophobic organic solvent,
[0010] (b) dissolving a stabilizer in water,
[0011] (c) admixing the solutions formed in step (a) and step (b)
to form an emulsion where the solution formed in step (b)
constitutes the continuous phase and the solution formed in step
(a) constitutes the disperse phase,
[0012] (d) evaporating the organic solvent to form a hardened
microsphere by polymer precipitation, from the double bond
functionalized polyhydric alcohol ester,
[0013] (e) recovering the microsphere.
[0014] In one embodiment, the double bond functionalized polyhydric
alcohol esters are obtained by polymerizing .epsilon.-caprolactone
monomer or a blend of .epsilon.-caprolactone and lactide monomer or
glycolide monomer in the presence of a polyhydric alcohol
containing from 3 to 6 hydroxyl groups to form a polyhydric alcohol
ester where the acyl groups contain free hydroxyl as their terminal
ends and reacting with maleic anhydride to convert some or each of
the free hydroxyls to moiety containing 2-carboxy ethenyl
group.
[0015] In another embodiment, denoted the second embodiment of the
invention, the invention is directed to a biodegradable injectable
microsphere having a mean transverse dimension ranging from about
15 to 60 .mu.m, formed of hardened double bond functionalized
polyhydric alcohol ester of polyester and loaded with from about 1
to 10% by weight of the microsphere of a drug or other biologically
active agent for sustained release after injection of the
microsphere into an animal. The release can take place over a
period ranging up to a few months.
[0016] The double bond functionality allows covalent bonding to
biologically active agents for delayed release as well as provides
the opportunity to form a hydrogel at the microsphere surface. This
provides the advantage of allowing two different release modes, one
from with the microsphere and the other from within the
hydrogel.
[0017] In one embodiment, the double bond functionalized polyhydric
alcohol ester is obtained by polymerizing .epsilon.-caprolactone
monomer in the presence of glycerol to form polyhydric alcohol
ester where the acyl groups contain free hydroxyl at their terminal
ends and reacting with maleic anhydride to convert some or each of
the free hydroxyls to a moiety containing a carboxy ethenyl
group.
[0018] In another embodiment, denoted the third embodiment of the
invention, amphiphilic plural block copolymers where one block is
from a polymer obtained by polymerizing .epsilon.-caprolatone
monomer in the presence of glycerol to provide esters with free
hydroxyl at the terminal ends of the acyl groups and reacting with
maleic anhydride to convert some or each of the free hydroxyls to a
moiety containing a 2-carboxy ethenyl group and the other block (s)
are from methoxy poly(ethylene glycol), are used to form loaded
microspheres by dissolving the plural block copolymer in a solvent
that does not dissolve the drug (or other bioactive agent) and
adding the drug (or other bioactive agent).
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows a diagram of the preparation of
poly(glycerol-co-caprolactone)maleic acid (PGCLM) microspheres with
entrapped protein ovalbumin.
DETAILED DESCRIPTION
[0020] The subject invention provides unique and advantageous
biodegradable injectable microspheres. The properties of the
microspheres of the subject invention can be modified as described
herein in order to give characteristics that are advantageous for
particular uses.
[0021] In one preferred embodiment, the surface of the microspheres
is hydrophilic. In another embodiment, the microspheres have a
hydrogel formed at the surface. Advantageously, the microspheres
can be utilized to deliver biologically active agents to a desired
site. Furthermore, the rate of release of the active agent(s) can
be carefully controlled as described herein.
[0022] A further advantage of the microspheres of the subject
invention pertains to the use of sucrose esters and dextran in
order to reduce toxicity associated with previously-known
microspheres.
[0023] In a preferred embodiment the microspheres are formed from a
double bond functionalized polyhydric alcohol ester of
polyester.
[0024] In one embodiment, the subject invention is directed to
forming a biodegradable injectable microsphere by a method
comprising the steps of:
[0025] (a) dissolving a double bond functionalized polyhydric
alcohol ester of polyester in a hydrophobic organic solvent,
[0026] (b) dissolving a stabilizer in water,
[0027] (c) admixing the solutions formed in step (a) and step (b)
to form an emulsion where the solution formed in step (b)
constitutes the continuous phase and the solution formed in step
(a) constitutes the disperse phase,
[0028] (d) evaporating the organic solvent to form a hardened
microsphere by polymer precipitation, from the double bond
functionalized polyhydric alcohol ester,
[0029] (e) recovering the microsphere.
[0030] In one embodiment, the double bond functionalized polyhydric
alcohol esters are obtained by polymerizing .epsilon.-caprolactone
monomer or a blend of .epsilon.-caprolactone and lactide monomer or
glycolide monomer in the presence of a polyhydric alcohol
containing from 3 to 6 hydroxyl groups to form a polyhydric alcohol
ester where the acyl groups contain free hydroxyl as their terminal
ends and reacting with maleic anhydride to convert some or each of
the free hydroxyls to moiety containing 2-carboxy ethenyl
group.
[0031] In another embodiment, denoted the second embodiment of the
invention, the invention is directed to a biodegradable injectable
microsphere having a mean transverse dimension ranging from about 5
to 200 .mu.m and, more specifically, 15 to 60 .mu.m, formed of
hardened double bond functionalized polyhydric alcohol ester of
polyester and loaded with from about 0.1 to 25% and, more
specifically, 1 to 10% by weight of the microsphere of a drug or
other biologically active agent for sustained release after
injection of the microsphere. The release can take place over a
period ranging up to a few months.
[0032] The double bond functionality allows covalent bonding to
biologically active agents for delayed release as well as provides
the opportunity to form a hydrogel at the microsphere surface. This
provides the advantage of allowing two different release modes, one
from with the microsphere and the other from within the
hydrogel.
[0033] In one embodiment, the double bond functionalized polyhydric
alcohol ester is obtained by polymerizing .epsilon.-caprolactone
monomer in the presence of glycerol to form polyhydric alcohol
ester where the acyl groups contain free hydroxyl at their terminal
ends and reacting with maleic anhydride to convert some or each of
the free hydroxyls to a moiety containing a carboxy ethenyl
group.
[0034] In another embodiment, denoted the third embodiment of the
invention, amphiphilic plural block copolymers where one block is
from polymer obtained by polymerizing .epsilon.-caprolactone
monomer in the presence of glycerol to provide esters with free
hydroxyl at the terminal ends of the acyl groups and reacting with
maleic anhydride to convert some or each of the free hydroxyls to a
moiety containing a 2-carboxy ethenyl group and other block (s) are
from methoxy poly(ethylene glycol), are used to form loaded
microspheres by dissolving the plural block copolymer in a solvent
that does not dissolve the drug (or other bioactive agent) and
adding the drug (or the bioactive agent).
[0035] The double bond functionalized polyhydric alcohol esters of
polyesters for step (a) include those described in Lang, M., et al,
Journal of Polymer Science: Part A: Polymer Chemistry, Col 40,
1127-1141 (2002) and can be synthesized as indicated therein.
[0036] Double bond functionalized polyhydric alcohol esters of
polyesters can be obtained by polymerizing .epsilon.-caprolactone
monomer in the presence of glycerol to provide esters with free
hydroxyl at terminal ends of the acyl groups and reacting with
maleic anhydride to convert some or each of the free hydroxyls to
moiety containing 2-carboxy ethenyl group. Examples of such
compounds and their preparation are described in U.S. Pat. No.
6,592,895 and may be referred to herein as PGCLM. The resulting
compounds have an average molecular weight, M.sub.n, ranging for
example, from 1,000 to 50,000.
[0037] Another embodiment is a double bond functionalized poly
(ortho esters) prepared by the reaction between the diketene acetal
3,9-diethylidene-2,4,8,10tetraoxaspiro[5.5]undecane and
1,10-decanediol (triethylene glycol can also be used to replace
1,10 decanediol). Molecular weights can be tailored by using
n-decanol to act as a chain regulator.
[0038] The poly (ortho esters) can be prepared by the addition of
diols triethylene glycol or 1,10-decanediol to
3,9-diethylidene-2,4,8,10 tetraoxaspiro[5.5]undecane. These
polymers can be prepared by dissolving the reagents in
tetra-hydrofuran and adding a trace of an acid catalyst.
[0039] A significant advantage of this polymer system is that the
nature of the diols controls the mechanical properties of the
polymer so that materials ranging from hard to soft flexible
materials can be made.
[0040] The solvent for step (a) is one that dissolves the double
bond functionalized polyhydric alcohol ester of polyester at room
temperature and that has a boiling point ranging, for example, from
30-45.degree. C. (which allows for easy removal of solvent). A
preferred solvent for step (a) is dichloromethane (bp of
38.9-40.degree. C.). Other suitable solvents for step (a) include
chloroform, ethyl acetate, and N,N-dimethylformamide.
[0041] For step (a), the double bond functionalized polyhydric
alcohol esters of polyesters are dissolved in the hydrophobic
organic solvent in an amount ranging, for example, from 0.5 to 10%
w/v. An increase in concentration causes an increase in mean
diameter of the microsphere ultimately obtained as well as in
loading efficiency of water soluble drug loaded as described
hereinafter and at least up to 6% w/v causes an increase in loading
level (drug %, w/w of microsphere).
[0042] The stabilizer for step (b) is a compound that is
essentially insoluble in the solvent of step (a), is removable by
washing with water, is stable in sunlight and artificial light,
reduces the interfacial tension between aqueous and organic phases
and limits collapse of droplets formed in step (c) before hardened
microspheres are obtained.
[0043] A preferred stabilizer is a sucrose ester for the
microencapsulation of PGCLM by the Solvent Evaporation Method. This
is a deviation from conventional microencapsulation processes using
the surfactant PVA solvent in the evaporation technique. PVA has
severe limitations due to its relative toxicity. Advantageously,
sucrose-based surfactants are biodegradable surfactants whose
hydrophilic and lipophilic properties can be adjusted by varying
fatty acid chain lengths. Examples of sucrose-based surfactants
include: TABLE-US-00001 *Sucrose Esters: Sucrose Monooleate Sucrose
Monolaurate Sucrose Mono-ester: 1 mole of fatty acid with 1 mole
sucrose Di-ester 2 moles of fatty acid Tri-ester 3 moles of fatty
acid
[0044] Other stabilizers include Pluronic F68 (ethylene
oxide/propylene oxide block copolymer having the structure:
HO(C.sub.2H.sub.4O).sub.a(C.sub.3H.sub.6O).sub.b(C.sub.2H.sub.4O).sub.aH
where a is 80 and b is 27, having a molecular weight ranging from
7680 to 9510 (and CAS Registry Number 9003-11-6), human serum
albumin (HSA), sodium chlorate, and charged and uncharged dextran
(weight average molecular weight ranging from 40,000 to
80,000).
[0045] Methods relevant to the practice of the subject invention
can be found at, for example, (Heller J. et al. (2002) "Injectable
Semi-Solid Poly (Ortho Esters) for the Controlled Delivery of
Therapeutic Agents: Synthesis and Application" Drug Delivery
Technology Vol. 2(1): 1-11 and Youan et al. (2003) "Evaluation of
Sucrose Esters as Alternative Sufactants in Microencapsulation of
Proteins by the Solvent Evaporation Method" AAPSPharSci Vol. 5(2):
1-7).
[0046] As noted above, a less preferred stabilizer is polyvinyl
alcohol (PVA) having a number average molecular weight ranging from
10,000 to 30,000 which is 85-90% hydrolyzed and is present in the
solution formed in step (b) in amount ranging from 0.5 to 10%,
w/v.
[0047] The volume ratio of solution formed in step (b) to solution
formed in step (a) admixed in step (c) can range, for example, from
3:1 to 10:1. Admixing can be carried out at 800 to 1,000 rpm for 5
minutes to 1 hours using a magnetic stirrer.
[0048] The evaporation of step (d) is readily carried out with
stirring while exposing the emulsion formed in step (c) to the
atmosphere while maintaining the emulsion at room temperature to
45.degree. C. Upon evaporation the microspheres precipitate and
become hardened because of the greater presence of stabilizer at
the surface of emulsion droplets.
[0049] The recovery of step (e) may be affected by centrifuging to
collect the microspheres, washing the microspheres with distilled
water to remove emulsifier, freeze drying and then storing until
used.
[0050] In one embodiment of the subject invention, the surface of a
microsphere is converted to a hydrogel. This is effected by
including a photoinitiator in the solution formed in step (a), e.g,
at a level of about 0.05 to 0.5% w/w of the double bond
functionalized polyhydric alcohol ester, e.g., 2,2-dimethoxy
2-phenyl acetophenone (DMPA) at a level of about 0.1% (w/w of
PGCLM), and then admixing the solutions formed in step (a) and step
(b) to form the emulsion of step (c) and causing cross linking at
the double bond functionality. This can be done by, for example, by
photocrosslinking, i.e., causing vinyl bonds to break and form
cross-links by the application of radiant energy. This can be done
by, for example, irradiating with a long wavelength UV lamp (365
nm, 16 watt) at room temperature while gently stirring overnight.
After that, the same hardened microsphere formation and collection
procedures can be used as when hydrogel is not formed at the
microsphere surface.
[0051] In a further embodiment of the subject invention, a drug or
other biologically active agent is loaded into the microspheres for
sustained release thereof. The drug or biologically active agent
can be, for example, a carrier of an aminoxyl radical or an
anti-inflammatory agent (e.g., serolimos) or an antiproliferative
drug (e.g., paclitaxel); a biologic; a protein; a cytokine; an
oligonucleotide including antisense oligonucleotide, or a gene; a
carbohydrate; hormone; etc.
[0052] When a water-soluble drug or other water-soluble
biologically active agent is to be loaded, this is readily carried
out by dissolving the drug or other agent in water to form an
aqueous solution that is admixed with the solution formed in step
(a) to form a water-in-oil emulsion that is admixed with the
solution formed in step (b) to form a water-in-oil-in-water
emulsion in step (c). In one embodiment, the water-soluble drug is
dissolved in water at a level of about 1-500 mg per ml and the
resulting solution is admixed with the solution of step (a) in a
volume ratio of about 1:3 to 1:30 (aqueous solution to solution of
step (a)) to form the water-in-oil emulsion.
[0053] Where an oil-soluble drug or other oil-soluble biologically
active agent is to be loaded, this can be accomplished by
dissolving the drug, or other agent, to be part of the solution of
step (a), e.g., at a level of 1 to 500 mg/ml and the resulting
solution is admixed with the solution formed in step (b) to form
the admixture of step (c).
[0054] In one embodiment, the injectable microspheres of the
subject invention can have a mean transverse dimension ranging from
about 20 .mu.m to about 50 .mu.m and can be loaded with from about
4% to about 8% by weight of the microsphere of drug or other
biologically active agent.
[0055] In one alternative, the surface of a PGCLM microsphere is
converted to a hydrogel as described above. In another alternative,
the surface of a PGCLM microsphere can be made more porous by
forming micropores therein, e.g., by using laser light from a laser
oscillator or by using an ultrasonic device to increase drug
release rate and microsphere degradation rate.
[0056] In all cases, double bonds at the surface of the microgels
can be reacted to covalently bond to a drug or other biologically
active agent. Loading efficiencies (actual drug loaded
(g)/theoretical load (g).times.100%) are readily obtained up to
about 45%, loading levels (actual loaded drug (g)/microsphere
weight (g).times.100%) are readily obtained up to about 8% and
cumulative release in 0.1M phosphate buffered saline (PBS) at
37.degree. is obtained up to about 50% over 50 days.
[0057] As indicated above, the injectable microspheres of the
subject invention including those where hydrogel is formed at the
microsphere surface, are biodegradable.
[0058] In the expression "some or each", "some" means more than one
and less than all, and "each" connotes all.
[0059] The term "biodegradable" is used herein to mean capable of
being broken down by various enzymes such as trypsins, lipases and
lysosomes in the normal functioning of the human body and living
organisms (e.g., bacteria) and/or water environment.
[0060] A third embodiment of the invention uses amphiphilic plural
block copolymers to form microspheres. One block can be from PGCLM
as described for the first embodiment. The other block(s) (which
are hydrophilic) can be from methoxy poly(ethylene glycol) (with
M.sub.n ranging from 300 to 8,000). The plural block copolymer is
prepared by reacting the terminal hydroxyl of the methoxy
poly(ethylene glycol) with one or more of the terminal carboxyls of
the PGCLM. To form drug loaded microspheres, the amphiphilic plural
block copolymer can be dissolved in a solvent for it, but not for
drug, to be loaded and then the drug is added, whereby drug loaded
micelle-like microspheres are formed, whereupon free drug and
solvent are removed followed by drying, e.g., freeze drying. A
suitable solvent for use in conjunction with a hydrophobic drug
such as taxol, is dimethylformarnide.
[0061] In one embodiment, the subject invention pertains to
terminal functionalized three-arm glycerol
poly(.epsilon.-caprolactone) microspheres (--OH, --COOH and
--C.dbd.C--) as the polymer matrix that provide a long-acting,
injectable drug delivery for sustained drug release over a period
ranging up to a few months. The functional groups of these
microspheres can be used for covalent binding of various bioactive
reagents by various activation methods, through hydrophilic active
chain end group --OH and --COOH as well as crosslinkable
--C.dbd.C-- to provide variable release profiles of, for example,
protein and to deliver the therapeutic agent efficiently.
[0062] Ovalbumin (OVA) is a non-toxic biodegradable protein from
chicken eggs, that has been successfully used in inducing antibody
(Ab) and cell-mediated immune (CMI) responses as well as for oral
vaccine delivery. In one embodiment, the subject inventions
provides OVA-loaded double-bond-carboxyl functionalized 3-arm
poly(glycerol-co-caprolactone)maleid acid (PGCLM),
hydroxyl-functional end group 3-arm poly(glycerol-co-caprolactone)
(PGCL), and network NPGCLM microspheres.These can be prepared by a
double emulsion technique (Price C, Stubbersfield R B, Kafrawy S E,
Kendall K D, Thermodynamics of micellization of
polystyrene-block-poly(ethylene/propylene)copolymers in decane,
Britic Polyco J 1989;21 :391-4).
[0063] FIG. 1 shows a diagram of the preparation of
poly(glycerol-co-caprolactone maleic acid (PGCLM) microspheres with
entrapped protein ovalbumin.
MATERIAL AND METHODS
Materials
[0064] Ovalbumin (albumin, chicken egg; Grade V), Maleic anhydride
(99%),2,2-dimethoxy 2-phenyl acetophenone (DMPA) and sodium dodecyl
suphate (SDS) were purchased from Sigma (St. Louis, Mo., USA).
Prestained SDS-PAGE standards (broad range from 14 to 94 kDa and
broad pl kit range from pH 3.50 to 9.50) were purchased from
Bio-Rad laboratories (CA, USA) as molecular weight and isoelectric
point makers. Polyvinyl alcohol (molecular weight, 12,000-23,000 of
87-89% hydrolyzed) (PVA), .epsilon.-caprolactone and glycerol
(99.0%) were purchased from Aldrich Chemical Co. (Milwaukee, Wis.,
USA). .epsilon.-Caprolactone was purified by drying with CaH.sub.2
for three days then distilled in vacuum at about 100.degree. C.
Glycerol was purified by distilling in vacuum. Chloroform
(Mallinckrodt Baker, Paris, Ky.) was extracted with water three
times to remove residual alcohol, dried with anhydrous MgSO.sub.4
overnight and distilled in an atmosphere of dry argon.
N,N-dimethylformamide (DMF) and triethylamine were obtained from
Aldrich Chemical (Milwaukee, Wis.) and used without further
purification. Osmium tetraoxide (2%) aqueous solution was purchased
from Electron Microscopy Science (Fort Washington, Pa.). All other
reagents were of analytical grade and used as received.
Preparation of PCL, PGCL and PGCLM
[0065] The preparation of the hydroxyl functionalized three-arm
poly(.epsilon.-caprolactone) PGCL and double-bond-functionalized
three-arm poly(.epsilon.-caprolactone) maleic acid (PGCLM) were
done according to prior published procedures (Shin I G, Kim S Y,
Lee Y M, Cho C S, Sung Y K, Methoxy polyethylene
Glycol)/s-caprolactone amphiphilic block copolymeric micelle
containing indomethacin: II. Micelle formation and drug release
behaviours, J Control Rel 1998;51:13-22). In brief, the hydroxyl
functionalized three arm poly(.epsilon.-caprolactone) (PGCL) were
synthesized by ring-opening polymerization of
.epsilon.-caprolactone (CL) in the presence of glycerol, which
acted as a core at the 20:1 feed molar ratio of CL to the hydroxyl
group of glycerol and stannous octoate (0.1 wt % of CL) in a
silinized Pyrox press reaction tube. After being vacuumed and
refilled with dry argon several times, the polymerization tube was
sealed in vacuum and placed in an oil bath at 130.degree. C. for 48
hours. The obtained polymer was dissolved in chloroform and then
gently poured into excess petroleum ether to precipitate the
product. The precipitates were washed with distilled water four
times and dried over P.sub.2O.sub.5 in vacuum at room temperature
until a constant weight was obtained.
[0066] Secondly, PGCL and 5 equivalents of the hydroxyl
functionality of maleic anhydride were placed in a three-necked
flask under a dry N.sub.2 environment and the flask was heated to
130.degree. C. for one day. The reaction mixture was then cooled to
room temperature and dissolved in chloroform. This chloroform
solution was poured into excess petroleum ether to precipitate
PGCLM. The powder precipitate was stirred in 500 mL of distilled
water for 4 h for the removal of any excess maleic anhydride. After
filtration, the precipitate was washed with distilled water four
times and dried over P.sub.2O.sub.5 in vacuum at room temperature
until a constant weight was obtained.
[0067] The linear high molecular weight PCL was also synthesized by
the same method in the absence of glycerol or any other alcohol as
an initiator, and the amount of stannous octoate was decreased to
0.05 wt % CL. The purpose of making high molecular weight PCL was
to provide controls for the PGCL and PGCLM characterization,
microspheres preparation and protein encapsulation.
[0068] Molecular weights (M.sub.n) of prepared polymers were
determined by gel permeation chromatography (GPC) using
tetrahydrofuran (THF) as the eluent (1.0 mL/min) with a Water 510
HPLC pump, a Water U6K injector, three PSS SDV columns (linear and
10.sup.4 and 100 .ANG.) in series, and a Milton ROM differential
refractometer. The sample concentration was 5-10 mg/mL of THF. The
columns were calibrated by polystyrene standards having a narrow
molecular weight distribution.
Preparation of Microspheres
[0069] PGCLM and NPGCLM microspheres with or without OVA were
prepared by the water-in-oil-in-water (w/o/w) emulsion techniques
(Price C, Stubbersfield R B, Kafrawy S E, Kendall K D,
Thermodynamics of micellization of
polystyrene-block-poly(ethylene/propylene) copolymers in decane,
Britic Polyco J 1989;21:391-4). In the case of PGCLM microsphere
preparation, 1 mL OVA aqueous solutions (containing 40, 80 or 170
mg of OVA) were first dispersed in a 10 mL PGCLM solution (4%, 6%,
8% w/v in dichloromethane) upon vigorous stirring (900 rpm for 15
min). The resulting w/o solution was then emulsified in a 50 mL
aqueous 1% PVA solution (w/v) for 30 min at 900 rpm. The resulting
w/o/w emulsion was gently stirred at room temperature (22.degree.
C.) by a magnetic stirrer (EYELA Magnetic stirrer RC-2) overnight
to evaporate the organic solvent. The resultant sample was
collected by centrifugation at 22.degree. C. (International
Centrifuges, Clinical Model, International Equipment Co. Needham
Hts, Mass. 02194 USA) and washed with distilled water at least four
times to remove the PVA emulsifier. The sample was freeze-dried for
3 days in a Virtis Freeze Drier (Gardiner, N.Y.) under vacuum at
-45.degree. C. to obtain the microspheres, which were stored in
vacuum desiccators at 4.degree. C. before characterization and
use.
[0070] To prepare microspheres which would have a crosslinked
surface network structure (NPGCLM), a photo-initiator, DMPA, of
0.1% (w/w) ofthe PGCLM was added to the initially formed w/o PGCLM
emulsion and then emulsified in 1% PVA solution to form w/o/w
solution. This w/o/w solution was then irradiated by a long
wavelength UV lamp (365 nm, 16 watt) at room temperature upon
gently stirring overnight. After that, the same collection
procedures as PGCLM microspheres preparation were used to collect
NPGCLM microspheres.
Microspheres Characterization
Fourier Transform Infrared (FTIR)
[0071] Fourier transform infrared (FTIR) spectra were obtained from
a Nicolet Magna-IR 560 spectrophotometer (Madison, Wis.). Two mg
PGCLM or NPGCLM microspheres were finely grinded with 15 mg KBr and
compressed into pellets for IR scanning in the range from 400 to
4000 cm.sup.-1 with a resolution of 8 cm.sup.-1 and carbon black
reference. The detector was purged carefully by clean dry helium
gas to increase the signal level and to reduce moisture
contamination.
Differential Scanning Calorimetric (DSC)
[0072] Differential scanning calorimetric (DSC) data were obtained
from a thermal analysis data system (DSC, 2920, TA Instrument). The
DSC was calibrated using indium as the standard. Samples (1-3 mg)
were heated in sealed aluminum pans from -110 to 190.degree. C. at
a scanning rate of 10.degree. C./min under nitrogen purge, with an
empty aluminum pan as a reference. The degree of crystallinity was
obtained from the first scan (10.degree. C./min), while the melting
temperature was from the second scan at 20.degree. C./min after
cooling from the first scan to RT. The heat of fusion
(.DELTA.H.sub.m) was determined by integrating the normalized area
of melting endotherms. The degree of crystallinity (X.sub.c) of the
synthesized polymer was then calculated according to the following
equation: Xc=(.DELTA.H.sub.m sample.DELTA.H.degree..sub.m, 100%
cystalline,).times.100%. where .DELTA.H.degree..sub.m is the
theoretical heat of fusion of PCL (135 J/g).
[0073] During the temperature range from -110 to 190.degree. C., no
T.sub.g was observed for the synthesized polymers.
Scanning Electron Microscopy (SEM)
[0074] The exterior and interior morphology of the PGCLM
microspheres was analyzed by scanning electron microscopy (SEM). In
brief, 5.0 mg of freeze-dried microspheres were dispersed in 500
.mu.L of distilled water, placed onto the top flat surface of metal
stubs and air-dried. The samples were sputtering coated (Edwards,
S150B, Sussex, UK) for 30 s and viewed under a STEREO SCAN 440
electron microscope (JEOL samples were LTD. TOKYO, JAPANO at 25
kV.
Backscattered Electron Imagines
[0075] Backscattered electron imagines with osmium tetraoxide
(OsO.sub.4) staining technique was used to qualitatively assess the
presence of the >C.dbd.C< functional groups on the surface of
PGCLM microspheres. Due to the attachment of OsO.sub.4 onto the
>C.dbd.C< bonds and hence their labeling by heavy element Os,
these double bond regions on the surface of the PGCLM microsphere
could be visually assessed by backscattered electrons for Os
element.
[0076] In brief, PGCLM microspheres were placed on aluminum stubs
by double-sided adhesive tapes. The mounted microspheres were
treated with osmium tetraoxide (2%) vapor for overnight in an
enclosed container for the attachment of OsO.sub.4 onto the
>C.dbd.C< bonds on PGCLM microsphere surface. After residual
OsO.sub.4 vapor was removed, the OsO.sub.4 treated microspheres
were then placed in a putter carbon coater (Edwards, Auto 306 High
Vacuum Evaporator, Edwards High Vacuum International, Wilmington,
Mass.) for 30 s to produce a carbon coating of approximately 150
.ANG. in thickness onto PGCLM microspheres. The coated microspheres
were viewed under a STEREO SCAN 440 (JEOL samples were LTD.TOKYO,
JAPAN) for backscattered electron imagines recording by a Tracor
Northern energy-dispersive X-ray analysis (Middleton, Wis.). The Os
peaks at 1.93 M.sub..alpha. and 8.91 L.sub..alpha. were used to
determine the presence of Os and hence >C.dbd.C< regions on
PGCLM microsphere surface.
Particle Size Analysis
[0077] The particle size and size distribution of the prepared
microspheres were measured by the laser light scattering method
(Brinkmann Particle Size Analyzer 2010, Brinkmann Instruments,
Inc., Cantiague Road, Westbury, N.Y. 11590). The dried microsphere
powder samples were first suspended in HPLC grade water (5-10%
vol.) and then slightly sonicated for achieving a homogeneous
suspension before size measurement. The data obtained included
volume density percentage, mean diameter and size distribution of
the microspheres.
Microsphere Encapsulation of Albumin and In Vitro Release
Encapsulation Efficiency Determinations
[0078] The amounts of OVA entrapped within PGCLM microspheres were
measured by dissolving 100 mg of the OVA loaded microspheres
prepared as described above in 2.0 mL of dichloromethane and the
OVA in the solution was extracted three times by 2.0 mL of
double-distilled water. The OVA content of the extraction solution
was determined by using a UV-Visible spectroscopy absorbance at 280
nm and a standard calibration curve from known concentrations of
OVA solutions. The OVA standard calibration curve is linear in the
1-100 .mu.g/mL concentration range with a correlation coefficient.
of 0.998. The amount of OVA encapsulated in the PGCLM or NPGCLM
microspheres, i.e. loading level in %, indicates the amount of OVA
in mg encapsulated per 100 mg of the microspheres. Also, the
loading efficiency of the process is defined as the ratio in % of
actually loaded OVA within 100 mg microspheres to the initial feed
OVA amount during the preparation of microspheres.
In vitro OVA Release Study
[0079] In vitro ovalbumin (OVA) release profiles were obtained by
incubating OVA-loaded microspheres (5 mg) in a centrifuge test tube
containing 5 mL of phosphate-buffered saline solution (pH 7.4) and
0.05% (w/v) of sodium azide used as preservative, at 37.degree. C.
At predetermined time intervals (50 days), the microsphere
suspension was centrifuged at 700 rpm for 10 min and the supemant
was collected. The concentration of OVA in the supernatant was
measured by a PERKIN Elmer Lambda 2 UVNIS spectrometer (Norwalk,
Conn.) at the wavelength 280 nm. Results were expressed as the
cumulative ovalbumin released percentage.
[0080] The test tube was replaced by fresh phosphate buffer to
maintain at a constant volume. Each experiment was conducted in
triplicate. To determine whether there would be any residual OVA
adhering onto the microsphere surface after centrifugation, a
control experiment was conducted by centrifuging a mixture of OVA
solution and OVA loaded microspheres according to the same
centrifugation condition above. The data of this control experiment
indicated negligible quantity of OVA residue remaining on the
microspheres surface.
Stability of OVA Encapsulated in Microspheres
[0081] The stability of OVA released from microspheres was analyzed
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of
OVA was qualitatively assessed by comparing with a standard OVA
marker according to the method of Laemmli (Kim S Y, Lee Y M,
Methoxy poly(ethylene glycol)/poly(s-caprolactone)amphiphilic block
copolymeric iiariosphere containing indomethacin: III.
Pharmacokinetic study in rat. J Contr Rcl. Submitted for
publication) . Briefly, the released OVA samples were dissolved in
a buffer containing 2% SDS, 0.5% .epsilon.-mercaptoethanol, 6%
sucrose, and 10 mM bromophenol blue. After the samples were boiled
for 5 minutes and cooled down on ice for 1-2 minutes, they were
loaded immediately in a 10% SDS-PAGE gel. The gel was run at 20-30
mA until the dye reached the front line. The gel was stained in
coomassie blue solution and destained until the proteins were
clearly seen. For each sample, two concentrations (2 .mu.g/lane and
4 .mu.g/lane) of OVA were used.
[0082] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0083] The following examples illustrate procedures for practicing
the invention. These examples should not be construed as limiting.
All percentages are by weight and all solvent mixture proportions
are by volume unless otherwise noted. It will be apparent to those
skilled in the art that the examples involve use of materials and
reagents that are commercially available from known sources, e.g.,
chemical supply houses, so no details are given respecting them
EXAMPLE 1
[0084] PGCL-Ma-3 solutions in dichloromethane were made up
containing 2% w/v polymer (denoted PGCLM21), 4% w/v polymer
(denoted PGCLM41), 6% w/v polymer (denoted PGCLM61) and 8% w/v
polymer (denoted PGCLM 81).
[0085] An ovalbumin protein (albumin, chicken egg, Grade V),
denoted OVA, was selected to represent drug to be loaded. It has
been used as an antigen in inducing antibody, cell-mediated immune
responses, as well as for oral vaccine delivery.
[0086] 1 ml OVA aqueous solutions containing 10, 40 or 85 mg OVA
were dispersed in 10 ml of PGCLM solution with vigorous stirring
(900 rpm for 15 minutes with a magnetic stirrer) to form a
water-in-oil emulsion where aqueous OVA solution was the disperse
phase in PGCLM solution continuous phase. The resulting w/o
emulsion was emulsified in 50 ml aqueous 1%
PVA(M.sub.n=12,000-23,009 87-89% hydrolyzed) solution (w/v) by
mixing for 30 minutes at 900 rpm with a magnetic stirrer to form a
w/o/w emulsion. The resulting w/o/w emulsion was gently stirred
overnight at 40% by a magnetic stirrer (EYELA Magnetic stirrer
RC-2) to evaporate the solvent leaving hardened microspheres loaded
with OVA, undissolved in the aqueous continuous phase.
[0087] The microspheres were collected by centrifugation at
22.degree. C. (International Centrifuge, Clinical Model,
International Equipment Co. Needham Hts, Mass. 02194 USA) and
washed with distilled water at least four times to remove PVA
emulsifier. The sample was then freeze-dried for 3 days in a Virtis
Freeze Drier (Gardiner, N.Y.) under vacuum at 45.degree. C. to
obtain the microspheres which were stored in vacuum dessicators at
40.degree. C. before characterization and use.
[0088] In another case, the procedure was the same as above but
DMPA at 0.1% (w/w of PGCLM) was added to the solution of PGCLM
before it was used to form the w/o emulsion with the aqueous
solution of OVA whereupon the w/o emulsion was admixed with the PVA
aqueous solution to form a w/o/w emulsion which was irradiated by
using a long wavelength UV lamp (365 nm, 16 watts) at room
temperature with gentle stirring overnight. After that, the same
procedure as used above, was used to collect the microspheres. The
result was cross-linked surface network structure microspheres
denoted NPGCLM.
[0089] Characteristics of OVA-loaded PGCLM and NPGCLM microspheres
are shown in the following tables. TABLE-US-00002 TABLE 1
Physicochemical characteristics of hydroxyl-functionalized
poly(glycerol-co-caprolactone) (PGCL) and
double-bond-functionalized poly(glycerol-co-caprolactone) maleic
acid (PGCLM) Polymers Mn T.sub.g T.sub.m .DELTA.Hm Xc Polymer
(kg/mol).sup.a (.degree. C.) (.degree. C.) (J/g) (%).sup.b
PGCL.sup.c 15.4 -- 52.5 61.3 45.4 PGCLM.sup.d 13.3 -- 48.9 64.8
48.0 NPGCLM 13.3 -- 51.3 47.3 35.0 PCL 56.9 -59 59 -- 52.sup.[28]
PCL-O 39.1 -54 52 -- 47.sup.[28] .sup.aDetermined by GPC with
polystyrene standards. .sup.bXc = (.DELTA.H.sub.m
sample/.DELTA.H.degree. .sub.m, 100% crystalline) .times. 100%.
.sup.cCL/OH is the molar ratio of CL monomer to hydroxyl and is
calculated by (mol of CL)/3 .times. mol of glycerol., 20/1
.sup.dPGCL-OH and 5 equiv of the hydroxyl functionality of maleic
anhydride molar ratio.
[0090] TABLE-US-00003 TABLE 2 Characteristics of OVA-loaded PGCL,
PGCLM and NPGCLM microspheres Loading Polymera OVA con. PVAb
DCM/H2O Mean OVA Efficiency Code (%, w/v) (mg) (%, w/v) (v/v) Diam.
( loading (%).sup.c PGCLM21 2 40 1 1/20 16.8 5.7 38.3 PGCLM41 4 40
1 1/20 18.0 6.0 41.1 PGCLM61 6 40 1 1/20 19.2 7.6 42.2 PGCLM81 8 40
1 1/20 21.0 4.1 43.2 PGCL61 6 40 1 1/20 34.3 7.2 36.2 NPGCLM61 6 40
1 1/20 21.9 4.2 45.3 .sup.aDichloromethane as solvent, PGCL Mn =
15,400; PGCLM Mn = 13,300; NPGCLM Mn = 13,200 .sup.bWater as
solvent .sup.cLoading efficiency = Actual load (g)/Theoretical load
(g) .times. 100%
[0091] The influence of OVA concentration on OVA loading
efficiencies is shown in Table 3. TABLE-US-00004 TABLE 3 Influence
of Ovalbumin concentration on OVA loading efficiency of
microspheres OVA Conc, Polymer OVA Mean Loading (mg/ml, Conc. PVA
loading Diameter Efficiency.sup.a Code H.sub.2O) (%, w/v) (%, w/v)
(%) (d.sub.vs, .mu.m) (% w/w) PGCLM61-1 10 6 1 1.4 17 43.0
PGCLM61-2 40 6 1 6.0 19.2 38.3 PGCLM61-3 85 6 1 8.1 16 28.7
.sup.aLoading efficiency = Actual load (%)/Theoretical load (%)
.times. 100%
[0092] The influence of PVA concentration on OVA loading
efficiencies is shown in Table 4. TABLE-US-00005 TABLE 4 Influence
of PVA concentration on OVA loading efficiency of microspheres OVA
Conc, Polymer OVA Mean Loading (mg/ml, Conc. PVA loading Diameter
Efficiency.sup.a Code H.sub.2O) (%, w/v) (%, w/v) (%) (d.sub.vs,
.mu.m) (% w/w) PGCLM61-4 80 6 0.5 1.4 26 32.7 PGCLM61-2 80 6 1 6.0
28.2 38.3 PGCLM61-5 80 6 5 7.4 24 26.7 .sup.aLoading efficiency =
Actual load (g)/Theoretical load (g) .times. 100%
[0093] The molecular weights and polydispersities are determined by
gel permeation chromatography using polystyrene standards. More
particularly molecular weights of prepared polymers (M.sub.n) are
determined by gel permeation chromatography (GPC) using
tetrahydrofuran (THF) as eluant (1.0 ml/min) with a Water 510 HPLC
pump, a Water U6K injector, three PSS SDV columns (linear and
10.sup.4 and 100 angstroms) in series, and a Milton ROM
differential refractometer, and the sample concentration is 5-10
mg/ml of THF and the columns are calibrated by polystyrene
standards having a narrow molecular weight distribution.
[0094] It should be understood that this example and embodiment
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
[0095] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0096] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
* * * * *