Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

Подождите немного. Документ загружается.

9.4 Medical Applications

505

FKJURI:

9.31. Spinal surgery (laminectomy) control material is to block formation of the massive adhe-

site in the rabbit showing massive fibrous adhesion sion (P. Glazer, surgeon; work supported by subcon-

(EF) to the dura mater (DM) that surrounds the tract from Bioelastics Research, Ltd.). (Reproduced

spinal cord (NC). The objective of the test elastic with permission from Alkalay et al.^'^

FIGURE

9.32. Spinal surgery (laminectomy) control

site in the rabbit showing massive fibrous adhesion

(EF) to the dura mater (DM) that surrounds the

spinal cord (NC). The objective of the test elastic

material, X-"-(GVGVP)25i (EM) is to block forma-

tion of the massive adhesion (EF) of Figure

9.31.

This

clearly occurs. (P. Glazer, surgeon; work supported

by subcontract from Bioelastics Research, Ltd.).

(Reproduced with permission from Alkalay et al.'^'^)

506 9. Advanced Materials for the Future

found attached to the dura; rather, the region is

filled by the elastic

matrix.

When the phase sep-

arated gel (G) state of (GVGVP)25i is extruded

into the laminectomy site adjacent to the dura,

as shown in Figure

9.33,

adhesion to the dura is

very limited.

Whether the

model

involves the

bloodied,

con-

taminated peritoneal

cavity

in the

rat,

strabismus

surgery in the rabbit eye, spinal surgery

(laminectomy) in the rabbit, or spinal surgery

(hemilaminectomy) in the goat, the elastic

protein-based

polymer

(GVGVP)n prevents

for-

mation of

debilitating

adhesions.

The decreased

quality of Life, the high health care costs due

to low back pain, addressed in section

9.1.5.1,

and the disability and medical care costs due

to the many other sites where postsurgical

and post-trauma adhesion occur combine to

position prevention of adhesions as the

potential "healer app" that could unleash the

greater potential of elastic protein-based

polymers.

9.4 A A Prevention of Urinary

Incontinence

The next aspect of soft tissue restoration

involves injectable implants of protein-based

polymers for the prevention of urinary inconti-

nence. We have been addressing this applica-

tion for a decade, first under the confidentiality

of a collaboration/license agreement with Bard

Urological Division and subsequently with the

support of the National Institutes of Health

by means of Phase I and Phase II SBIRs. It

has been during work on this application that

the essential developments of purification, as

demonstrated by Figure

9.14,

and stimulation of

differentiated tissue generation have been

achieved (as shown in Figure 9.35, below).

Accordingly, with the magnitude of the health

care problem noted in section 9.1.5.2, this also

becomes a reasonable candidate for the "healer

app"

with which to unlock the potential of

protein-based polymers as medical devices.

FIGURE

9.33. Spinal surgery (laminectomy) test site

in the rabbit model showing the test elastic material,

the gel state of (GVGVP)25i (G), to have blocked

formation of the massive fibrous adhesion (EF) to

the dura mater (DM) that surrounds the spinal cord

(NC) (P. Glazer, surgeon; work supported by sub-

contract from Bioelastics Research, Ltd.). (Repro-

duced with permission from Alkalay et al.^*^)

9.4 Medical Applications 507

Reasons for this optimism are many. They

include a cost per gram of one-thousandth that

of Contigen® (an FDA-approved material to

correct urinary incontinence), the capacity to

purify to the exacting levels required for use as

an injectable implant, and the demonstration

that injectable implants of certain compositions

induce differentiated generation of new tissue,

whereas Contigen® induces formation of dense

scar tissue and is of limited success with a 10%

to 15% cure rate at 3 years. Collagen injections

cause a mild inflammatory response with the

result of scar tissue formation. The contracture

that scar tissue undergoes and the wisdom of

repeating the procedure with more scar tissue

formation would seem to limit the potential of

collagen injections. Also there exists the fear-

some possibiUty of prions in collagen obtained

from animal sources (e.g., mad cow disease as

considered in Chapter 7). Even so, Contigen®

did cover new ground in obtaining FDA

approval for medical use of protein-based

materials.

9.4.4.4.1 Possibility of Elastic Protein-based

Polymers as Inert Space Fillers

The objective in overcoming urinary inconti-

nence is to introduce a material at the base of

the bladder to provide a volume for physical

support. This has been attempted in the past

with inert materials. In fact, such an approach

could be used with (GVGVP)25i by extrusion

through a hypodermic needle to fill volume at

the base of the bladder. This has been demon-

strated with the present animal model of sub-

cutaneous injection in the guinea

pig.

As shown

in Figure 9.34, a 30mg subcutaneous injection

of (GVGVP)25i results in a volume filled with

the phase separated state of (GVGVP)25i and

surrounded by a fine fibrous capsule, the fine

fibrous capsule that results from minor residual

impurities arising from production by E. coli.

Here the fibrous capsule serves to hold the

material in place and thereby to limit migra-

tion. This simple approach could itself be

useful, but it does not bring out the full poten-

tial of elastic protein-based polymers for this

apphcation.

FIGURE 9.34. Subcutaneous site in the guinea pig for

injection of the purified gel (GVGVP)25i, showing

a fine fibrous capsule due to a small impurity to

limit migration of the tissue volume augmentation.

(Reproduced with permission from Urry et al.^^)

9.4.4.4.2 Injection of Protein-based Polymer

Composition That Induces Tissue Generation

for Support of the Urinary Bladder

Our primary objective is to achieve augmenta-

tion with development of a natural, long-lived

tissue. The generation of elastic fibers would be

expected to result in a long-lived augmentation,

because elastic fibers in the body have half-lives

on the order of 60 years or more. Collagen, on

the other hand, turns over every 9 to 12 months.

As shown in Figure 9.35, subcutaneous injec-

tion of 30 mg of [(GVGVP)io-GVGVP-

GRGDSP-GVGVP)io]i8(GVGVP) results in a

508

9. Advanced Materials for the Future

4' '• • !

, -'?*•

•. V

•. ••"•!• •'•'•' .••'<v^;'-^'->-..•••• •

FIGURE

9.35. Subcutaneous site in the guinea pig

2 weeks after a 30mg injection of a highly purified

elastic protein material with a GRGDSP cell

attachment site, namely, [(GVGVP)ioGVGVP-

GRGDSP(GVGVP)io]7(GVGVP). (A) Low magni-

fication showing much tissue generation with

angiogenesis and some remaining polymer seen as

vesicles. (B) Higher magnification showing erythro-

cytes in capillaries and spindle-shaped fibroblastic

cells presumably synthesizing collagen and elastin

fibers. (Reproduced with permission from Urry

et al.^«)

tissue generation with what appears to be a

natural distribution of collagen and elastic

fibers. In particular, the generation of elastic

fibers would prevent contracture and would be

expected to result in a long lasting tissue gen-

eration, because the half-life for elastic fiber in

the human is of the order of a lifetime.

9.4.4.5 Plastic Surgery to Improve Body

Contours Formed by Soft Tissues

A composition could be injected to induce

tissue generation for improving body contour.

Wrinkle removal provides the simplest exten-

sion of tissue augmentation or generation, but

the concept could be applied at many sites

where soft tissue generation may be considered

a desirable result.

9.4.4.6 Temporary Functional Scaffoldings

for Soft Tissue Restoration

Whenever a dynamic structure fails or is so dis-

eased that it needs to be replaced, the concept

of temporary functional scaffoldings comes into

play. Examples are synthetic arteries, urinary

bladder, intervertebral discs, and even a tem-

porary functional scaffolding for skin replace-

ment, for example, after burns.

Each of these structures begins with a struc-

tural integrity and a dynamic functional range

that becomes lost through any number of

disease processes. In each of these cases and

many others, the concept of a temporary func-

tional scaffolding couples with the remodeling

capacity of natural cells sensing the demands of

a normal tissue environment to result in tissue

9.4 Medical Applications

509

restoration. An explicit example of the urinary

bladder follows.

The concept of using temporary functional

(elastic) scaffoldings in soft tissue restora-

tion has been tested in a simulated urinary

bladder model. An explant from human ureter

was placed on a large disk of cross-linked

matrix of [(GVGVP)io-GVGVPGRGDSP-

GVGVP)io]i8(GVGVP), and the outgrowth

of human uroepithelial cells was verified.

This matrix closely matched the elastic

modulus (compUance) of the natural human

bladder.

This disk, with a human ureteral explant posi-

tioned on it, was placed on the mandril of a sim-

ulated bladder apparatus, as shown in Figure

9.36. The apparatus could be filled with culture

medium and held in a static nonextended state

for several days. Alternatively, the simulated

bladder could be slowly filled to 100% exten-

sion over a 3-hour period and emptied in 25

seconds.

This filling and emptying continued for

several days.

The objective becomes the comparison of the

outgrowths of human uroepithelial cells from

the explants as a function of static and dynamic

conditioning. Shown in Figure 9.37A is the

static case where a careful examination allows

delineation of the edges of the outgrowth

from the black area where the nontransparent

explant resides. Figure 9.37B demonstrates the

result from the dynamic case that simulated

the natural filling and emptying of a urinary

bladder. The tissue outgrowth in the dynamic

case is much richer, exhibiting a higher cell

density and a thicker extracellular matrix. Sub-

jecting the outgrowth of human uroepithelial

cells from a ureteral explant to the force changes

that occur naturally during bladder function

stimulate the cells to begin developing the appro-

priate bladder tissue. Thus, Figure 9.37 provides

an example of the concept of designed tempo-

rary elastic scaffoldings containing cell attach-

ment sequences that, when combined with the

mechano-chemical transductional properties of

cells,

can result in tissue restoration.

FIGURE

9.36. Disassembled Simulated Bladder

Chamber. (A) Mandril covered by a large disk

of elastic protein-based polymer membrane,

X''-[(GVGVP)ioGVGVPGRGDSP(GVGVP)io]7(G

VGVP), containing GRGDSP cell attachment

sequences. Human ureteral explant is centrally

placed on the elastic disk. (B) Top chamber to be

placed over the mandril with filling/emptying port

and a window for microscopic viewing of outgrowth

from explant. (Unpublished results^^^)

510

9. Advanced Materials for the Future

FKJURI:

9.37. Oulgrowlh from human ureteral

explanl on elastic protein-based polymer membrane,

X^"-[(GVGVP),oGVGVPGRGDSP(GVGVP),„]7(G

VGVP), during a several-day period in simulated

urinary bladder. (A) Static circumstance with no

filling and emptying gave good outgrowth compared

with collagen-covered substrate. (B) Dynamic filling

(3 hours) and emptying (25 seconds) resulted in

much more dense outgrowth of cells and thicker

extracellular matrix. (Reproduced with permission

from Urry and Pattanaik et al.'-'*)

9.4.5 Controlled Release of

Pharmaceuticals by Tftype

(Transductional) Protein-based

Polymers

The special energy conversion (transductional)

capacities designable into elastic protein-

based materials provide unique opportunities

for a remarkable range of systems capable of

controlled delivery of drugs and other more

complex therapeutic molecular constructs.

Each of the 18 pairwise energy conversions,

described in Chapter 5 for which elastic

protein-based materials can be designed, can be

employed to control release of pharmaceuti-

cals.

Basically the elasticity, the inverse temper-

ature transitional behavior, and knowledge of

the forces responsible for that behavior provide

an unprecedented potential for the develop-

ment of controlled release systems. It is not an

issue of using a single or even several composi-

tions,

and it is not primarily an issue of working

with the different physical states of these com-

positions to coerce a more useful state for a

9.4 Medical Applications

511

particular controlled release problem. Instead,

the composition of the bioelastic material

should be designed specifically to have the

optimal properties perceived for the particular

controlled release problem at hand.

Again we recall the statement of Bronowski,^

"/n ejfect, the modern problem is no longer to

design a structure from the materials (available)

but to design materials for a structure.'' The

chemical nature of the pharmaceutical to be

delivered and the preferred circumstance for its

delivery determine the structure of the elastic

protein-based polymer to be designed for that

purpose. The approach is emphatically not one

of using an "off-the-shelf" (available) polymer

and coercing it chemically and physically to

approach a desired release. The potential appU-

cations and structures of bioelastic materials in

the area of controlled release can be expected

to be as numerous as the pharmaceuticals and

circumstances to be considered and to include

the less exotic application of nonadherent drug-

delivering wound coverings.

9.4,5.1 Positively Charged Drugs

Delivered by Negatively Charged

Polymers: Consideration of Analgesics,

Anesthetics, and Endorphins

9.4.5.1.1 The Chemical Nature of Analgesics

and Anesthetics

Most of the analgesics and anesthetics used

today are tertiary (or secondary) amines, which

at physiological pH are positively charged.

These include alfentanil, bupivacaine, butor-

phanol, chloroprocaine, cocaine, codeine, dyclo-

nine,

fentanyl, ketamine, lidocaine, meperidine,

mepivacaine, morphine, nalbuphine, prilocaine,

sufentanil, and tetracaine. There are also, for

example, the intravenous anesthetics Brevital

(methohexital sodium) and Diprivan (propo-

fol) and the topical analgesic Zostrix (cap-

saicin) that have the capacity to contain

negatively charged, for example, phenolate,

species. In addition to being able to carry a

charge, these drugs all exhibit substantial

hydrophobic character, that is, they contain

both polar (ionizable) and nonpolar moieties.

These are exactly the types of molecular

species, the sustained release of which can be

most effectively achieved by the transductional

protein-based polymers under consideration

here.

9.4.5.1.2 The Chemical Nature of Endorphins

The term endorphin is an ehsion for "end-

ogenously produced morp/zmelike substance,"

and it has come to stand for the set of end-

ogenous peptides that includes the enkephahns,

dynorphins, endorphins, and their synthetic

analogues, which are also collectively referred

to as opioid peptides.^^'^^ Great excitement

occurred more than two decades ago with the

discovery of enkephalins by Hughes and

coworkers.^^ Endorphins are the body's own

means of alleviating pain. This introduced the

possibility of pain relief without development

of debihtating cravings or addictions. The real-

ization of opioid peptides as pharmaceuticals,

however, has been limited in significant part

due to their rapid in vivo metabolism,^^ to their

ehciting serious side effects at higher concen-

trations, and to the problem of delivery at con-

stant and competent doses to their sites of

activity whether in the central nervous system

or at peripheral sites.^^'^'^'^^

Much work has been directed toward the

preparation of opioid peptide analogues to alle-

viate the problem of proteolytic inactivation,

to define the several receptors,^^'^^ to achieve

receptor selectivity and elicit selective activities

thereby minimizing side effects, and generally

to understand the complex mechanisms

involved in substance abuse.^^ For that work

to be more fully utilized in health care and in

alleviating suffering, delivery processes are

required to protect the peptide from prote-

olytic degradation, to access various sites in the

body,^^'^ and to provide for the desired release

profiles. This problem provides an opportunity

for elastic transductional protein-based poly-

mers to fill a much-needed role.

9.4.5.1.3 Generally Considered Means of

Controlled Release by Polymeric Systems

The development of polymers as controlled

release vehicles has been of long-standing

interest.^^"^^ Kost and Langer^"^ consider four

512 9. Advanced Materials for the Future

different controlled release systems for poly-

meric hydrogels: (1) diffusion controlled, (2)

chemically controlled, (3) solvent controlled,

and (4) release induced by external factors. Pitt

et al.^^ and Peppas and Korsmeyer^^ list similar

mechanisms. TTiere is another category of

mechanism that stands in its own right and

that encompasses all of the above. It utilizes

the inverse temperature transition and may be

called the Trtype

transductional

mechanism of

general development in this volume.

9.4.5.1.4 The Opportunity for Elastic

Transductional Protein-based Polymers

as Controlled Release Vehicles

Biocompatible and biodegradable polymers

provide the potential for the delivery of

analgesics and anesthetics in an anatomically

localized manner and at sustained optimal dose

levels for alleviating pain. These controlled

release vehicles would eliminate the occurrence

of peak concentrations during which times toxic

reactions are more likely to occur. Transduc-

tional elastic protein-based polymers hold

promise for sustained and even constant (zero

order) release levels of analgesics and anesthet-

ics that would help to alleviate untoward clini-

cal responses. In fact, as discussed below,

transductional protein-based polymers have the

demonstrated capacity to release a cationic

morphine analogue and a cationic opioid

peptide at a constant level for a given surface

area and for periods up to many months. Fur-

thermore, they can be designed to release at pre-

ferred levels and time periods and to be formed

into useful drug delivery

vehicles:

injectable vis-

coelastic implants, injectable nanoparticles and

microspheres, trocar implanted elastic rods,

laparoscope implanted sheets, and patches for

transdermal delivery.

9.4.5.1.5 Zero Order Release of Positively

Charged Leu-enkephalin Amide from

Negatively Charged Polymers

Leu-enkephalin amide (H^-Tyr-Gly-Gly-

Phe-Leu-NH2, LEA^) is a positively charged

opioid peptide from the endorphins that are so

well known for causing the "runner's high."

When added to a solution containing a rela-

tively hydrophobic carboxylate-containing

elastic protein-based polymer, LEA^ induces

phase separation. The positively charged drug

ion pairs with the elastic protein-based polymer

and lowers Tt, the onset temperature for the

inverse temperature transition. In this manner

the drug provides the chemical energy for its

own packaging into the drug delivery

device.

the glutamic acid (Glu, E)-containing polymer

becomes more hydrophobic by replacement

of less hydrophobic valine (Val, V) residues

by more hydrophobic phenylalanine (Phe, F)

residues, there occurs an increase in pKa, and

there results an increase in binding affinity of

drug to polymer in the phase separated state.

In the example to be discussed, loading

occurred with a 50% excess of drug over car-

boxylate ion pairing sites. The Leu-enkephalin

amide release profiles in Figure 9.38 were

obtained using a cylindrical tube with a shallow

conical bottom, as also depicted in Figure

9.38B, and replacing the overlying buffer solu-

tion every 24

hours.

^°

Note that the drug deliv-

ery vehicle disperses as the drug is released,

because, in fact, the drug provides the glue that

holds the drug delivery vehicle together. The

excess drug is released during the first 12 days.

Once this has occurred, there follows a remark-

ably constant release, which continues until the

sample is totally dispersed.

For 250 mg of poly(GVGFP GEGFP

GVGVP), zero order release occurs at the level

4.7

|Lim/day

for approximately 1 month, and

the turn off is quite abrupt as the materials

come from the bottom conical part of the tube.

For 250 mg of the more hydrophobic protein-

based polymer poly(GEGVP GVGVP GVGFP

GFGFP GVGVP GVGVP), zero order release

is maintained constant for over 1 month at

about 2.7|im/day and again turns off abruptly.

For 250 mg of the most hydrophobic elastic

protein-based polymer, poly(GFGFP GEGFP

GFGFP), of the series, zero order release

is maintained constant for nearly a period

of 3 months at the level of

1.9|im/day

and

again shuts off abruptly. Of course, the initial

burst can be eliminated by not loading with an

excess of drug, but an initial excess may be

helpful in vivo to more quickly establish a

steady state.

9.4 Medical Applications

513

Release Profiles of Leu-enkephaline amide from

Glu-containing Protein-based Polymers

10.0

7.5

•S

5.0

2.5 H

0.0

250 mg polymer

conditions:

pH 6.8

° 1.5 equivalents of drug for each

Glu binding site

polyCGVGFP GEGFP GVGVP)

4.7

mole

days

30 40

10.0

7.5-^

5.0

2.5

0.0

10.0-

250 mg polymer

conditions:

pH 6.8

1.5 equivalents of

drug

for each

J Glu binding site

1 DQoa'-'Qoaaaaoaaa

I.I.I.

poly(GEGVP GVGVP GVGFP

GFGFP GVGVP GVGVP)

u J

'°°°°°°°°°°°oaaa --- mole

°°a

1 . 1 . 1

20 30

days

7.5'

5.0

2.5

0.0

250 mg polymer

conditions:

pH 6.8

1.5 equivalents of drug for each

Glu binding site

poly(GFGFP GEGFP GFGFP)

•••^

•*********»»****^^»»<

1.85

mole

20 40 60

100

days

FIGURE

9.38. Release of positively charged Leu- release levels are maintained constant out to 3

enkephalin amide from designed, negatively charged months. Initial burst release due to 50% excess drug

polymers with systematically increased hydropho- over polymer sites. (Reproduced with permission

bicity and pKa shifts. For constant surface areas, from Urry et al.^°)

514

9. Advanced Materials for the Future

It is quite clear that the ion-pairing approach

using hydrophobic-induced, pKa-shifted,

anionic sites within polymers capable of inverse

temperature transitions provides a means of

achieving constant release of positively charged

drugs over substantial periods of time. As

discussed below, similar results have been

obtained for the narcotic antagonist naltrexone.

9.4.5.1.6 Drug Addiction Intervention:

Zero Order Release of Positively Charged

Naltrexone from Negatively

Charged Polymers

The approach considered for drug addiction

intervention is to treat the addict, after

withdrawal, to prevent recurrence of active

dependency. An effective drug is the narcotic

antagonist naltrexone. Two nanograms per mil-

Uliter of this drug in the blood plasma can block

the action of a

mg dose of heroin and deter

recurrence of active dependency.

The limitation with the use of this drug arises

from the problem of patient compUance. If for

whatever reason the patient feels compelled

again to have the high, the medication is dis-

continued and the addict again becomes

actively dependent. One way to overcome this

problem is a subcutaneous implant of a con-

trolled release device that would maintain an

effective range of drug release for sufficient

periods to get the addict beyond the return to

active dependency. This could be a device that

releases competent doses for 1 month or more.

Naltrexone is a positively charged drug.

Therefore, as for the opioid peptide Leu-

enkephalin amide discussed above, the design of

the bioelastic drug delivery device is one of neg-

atively charged polymers; the two polymers in

Figure 9.38B,C will be discussed for ease of

comparison. The release profiles are contained

in Figure 9.39. For 250 mg of poly(GEGVP

GVGVP GVGFP GFGFP GVGVP GVGVP)

and a 50% excess of naltrexone over the number

of carboxylates, zero order release is maintained

constant for over 1 month at 2.7 |Lim/day (the

same as for Leu-enkephalin amide) and, of

course, turns off abruptly, just as occurred with

Leu-enkephalin amide. For

250

mg of the more

hydrophobic elastic protein-based polymer

poly(GFGFP GEGFP GFGFP), zero order

release of naltrexone continues constant for

Naltrexone Release Profile from Glu-containing Protein-based Polymers

8.0

6.0-

4.0 H

250 mg polymer

loading conditions: pH 7.4

1.5 equivalents of

drug

for each

Glu binding site

poly(GEGVP

GVGVP GVGFP

GFGFP GVGVP

GVGVP)

poly(GFGFP

GEGFP GFGFP

>.e

2.2 mole

2.7 mole

100

FIGURE 9.39. Release of the positively charged nar- levels are maintained constant out to 3 months.

CO tic antagonist naltrexone from negatively charged Initial burst release due to 50% excess drug over

polymers with systematic increases in hydrophobic- polymer sites. (Courtesy Bioelastics Research, Ltd.)

ity and pKa shifts. For constant surface areas, release