Biocompatibility of N-(2-hydroxypropyl) methacrylamide copolymers containing adriamycin

Biocompatibility ofN-(2-hydroxypropyl) methacrylamidecopol~ers cons adriamycin ~~o~e~~i~, and effect on haema~poie~c stem cells in bone m~row in I&O and mouse splenocytesand human peripheral bloodlymphocytesIZIvitro. B. nova, Instituteof

M. Silej and V. Vegan

Microbiology, Czechoslovak Academy of Sciences, CS- 142 20 Prague, Czechoslovakia

K. Ulbrich,J. Strohalmand J. Kopecek fnstitute of Macromolecular

Chemistry. Czechosfovak Academy of Sciences, CS- 16206

Prague, Czechosfovakia

R. Duncan Cancer Research Campaign Polymeric-Controlled Drug Delivery Group, Department of Keele, Staffordshire, ST5 5BG, UK (Received 1 February 1988; accepted 1 May 1988)

of Biological Sciences, University

#“(Z-hydroxypropyl)methac~lamide polymeric prodrugs containing adriamycin bound to polymers via glycylphenylalanyllaucylglycine side chains and, in one case, galactosamine bound via the same sequence, were tested for immunogenicity after intravenous, subcutaneous and oral application in two inbred strains of mice. The serum antibody level was determined by enzyme-linked immunoassay on the 3rd and 6th day after the last treatment. It was found that antibodies were only produced in very small amounts. In some experimental groups, the antibody titres measu~d following administration of copolymer conjugate were comparable with those present in non-treated controls. Attachment of adriamycin to N-(Z-hydroxypropyl)methacrylamide copolymer considerably decreased its toxicity against haematopoietic precursors in bone marrow as measured by the in wivo colony-forming unit-spleen assay and its ability to inhibit [3H] thymidine incorporation by mouse splenocytes and human peripheral blood lymphocytes measured in vitro. Keywords: B;ocompatibflity, copofymers, antibiotics

N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers have been studied as possible carriers for drugs and targeting moieties in affinity therapyle3. They can be synthesized to contain oligopeptide side chains terminating in reactivep-nitrophenylestergroups, which allow binding to biologically active compounds containing amino groups using an aminolysis reaction. It is possible to synthetize oligopeptide side chains which guarantee stability of the bond between the drug and the polymer in the bloodstream, but are susceptible to bi~egradation by intracellular lysozomal enzymes4-6. Biodegradabili~ is a prerequisite for the

pharmacological

activity

of the

preparation”7.

Such

conjugates have been called HPMA polymeric prodrugs’ and they have been studied as potential cancerostatics*.’ and immunomoduiators7, the targeting either

moiety

lo, “,

attached.

galactosamine’2.‘3

their

activity

For delivery

depending

on

to cancer tissue,

or fucosylamine8.9

have

been

used as targeting residues conferring specificity for hepatoma or mouse L12 10 leukaemia, respectively. For targeting to lymphatic tissue, anti-Thy 1 .2 or anti-la antibodies have been attached to the drug-polymer conjugate7, “, “. Anthracycline antibiotics, daunomy~in and adriamycin (ADR), or a protein synthesis inhibitor, puromycin, have been bound to HPMA copolymers as the pharmacologically active agent. Such

__l__-_

drug-copolymer

Correspondence to Dr 6. Rihova, Institute of Microbiology, Videnska 1083, 142 20 Prague 4, Czechoslovakta.

L 12 1 O8 and to splenocytes and thymocytes”. ’ ’ in vitro and in vivo they were able to prolong life and produce an increase

(0 1989

Butterworth Et Cc (Publlshersj Ltd. 0142-961

conjugates

are cytotoxic to mouse leukaemia

Z/89./050335-08$03.00 Blomatenals

1989, Vol IO July

335

BiocompetibiMy of HPMA copolymer conjugates: B. Ribova et a/

Table 1

Cbaracterizalion of the polymers

Polymer

Structure

Content of side chains (mol%)

MW

Bound ADR wt%

Precursor 7 Precursor 2 Sample 1

P-Gly-Phe-Leu-Gly~Np P-Gly-Phe-Leu-Gly-ONp P-Gly-Phe-Leu-Gly-AOR ,,Gly-Phe-Leu-Gly-ADR P ‘GlyPhe-Leu-Glygalactosamine

3.8 7.8 2.5 2.4

24 000 19000 24 000

8.5

19000

7.3

Sample 2

4.0

in the number of long-term survivors when tested in L 12 10 leukaemia bearing DBAp mice“’ and effectively suppress antibody response7, lo, ’ ’ when used as immunomodulators. Conjugation of anthracyclines to the polymer considerably reduced the non-specific toxicity of the drug as judged by toxicity in liver, heart and bone marrow stem cells’. Previously, we have shown that HPMAcopolymers are very weak thymus-independent immunogens and that the intensity of the antibody response elicited depends on many factors, including copolymer molecular weight and composition of oligopeptidic side chains and of the genetic background of the immunized animals’4-‘6. Following immunization with soluble or complete Freund’s adjuvans (CFA) incorporated HPMA copolymers, different inbred strains of mice respond with a low IgM antibody response. HPMA copolymers activate neither the classic nor the alternative complement activation pathway”. If the molecular weight is below 45 kD, they are eliminated from the organism within hours by urinary excretion” and do not accumulate in any tissue. The biocompatibility of HPMA copolymers developed as polymeric prodrugs is of fundamental importance to their possible therapeutic use and therefore requires systematic testing. In this study, we have chosen to focus on one particular aspect of the biocompatibility of two HPMA copolymer conjugates. Both contained ADR bound to the polymer backbone via glycylphenylalanylleuc~glycine side chain and one contained in addition galactosamine (bound via the same peptide sequence (Table 1). The ability of these polymers to induce an antibody response was investigated using the enzyme-linked immunoassay (ELiSA) to measure antibody levels in mouse serum. In addition, the toxicity of the applied polymers against haematopoietic precursor cells in bone marrow was studied by taking both femurs from immunized mice, extracting the bone marrow and subsequently measuring the number of colony-forming unitspleens (CFU-s) in recipient irradiated mice. Finally, the effect of HPMA copolymers containing ADR on the proliferation of mouse splenocytes and human peripheral lymphocytes in vitro was tested by measuring the incorporation of [3H]thymidine in the presence and absence of concanavalin A (Con A).

MATERIALS

AND METHODS

Chemicals 1-aminopropan-2-01, methacryloylchloride, dicyclohexylcarbodiimide, dimethylsulphoxide (DMSO) and 4-nitrophenol were from Fluka AG, Buchs, Switzerland. Glycylphenylalanine. leucylglycine, gatactosamine and ADR were from Sigma Chemical Co., Poole, Dorset, UK.

336

was prepared, as previously described’g,

Biomaterials

Preparation of MA-Gly-Phe-Leu-G&-OH. H-Leu-Gly-OH (0.011 M) and NaHCOs (0.02 16 M) weredissolved in 50 ml of water and this solution added to a solution of 0.01 M N-methacryloylglycylphenylalanyl p-nitrophenyl ester in 70 ml of dioxan**. The reaction mixture was stirred for 48 h at r.t., dioxan evaporated and after filtration the solution was acidified by diluted HCI (1: 1) to pH -2. The mixture was immediately extracted three times with 40 ml of ethyl acetate and the solution dried using Na,SO,. Ethylacetate was evaporated off and oily product was rubbed to powder in diethylether. White crystals obtained were washed with diethylether and dried in vacua. Yield 74%. m.p. 148-l 50°C. Preparation of MA-G/y-Phe-Leu-G/y-O~p. 0.0265 ~1 of MA-Gly-Phe-Leu-Giy-OH was dissolved in 200 ml of dry tetrahydrofuran (THF) and the solution cooled to 0°C. Solutions of 0.029 1 M of dicyclohexylcarbodiimide in 20 ml THF and 0.0291 M p-nitrophenol in 15 ml THF were than added whilst cooling and stirring continuously. After 2 h, the reaction mixture was kept for 24 h in a refrigerator (4°C). The dicyclohexylurea formed was filtered off and THF evaporated. Crude crystals were dissolved in 200 ml of ethylacetate, after cooling to 0°C this solution was filtered and evaporated to 80 ml. After precipitation into one litre of diethylether, the product was isolated and was further purified by crystallization from the mixture of acetondiethylether (3: 7). Yield 60%, m.p. 175-l 77°C && = 9300 (I mol-’ cm-‘) TLC:& = 0.50 (aceton-diethylether 3:7). Analysis for

C

1989, Vol f0 July

Meth-

H

N

&HaaN& Calculated Found

59.89 59.78

6.07 6.18

12.04 11.68

Preparation of reactive polymeric precursors Polymeric precursors were prepared by radical precipitation co~lymerization of HPMAand MA-Gly-Phe-Leu-Gly-ONp in acetone as previously described”. Molecular weights of polymers were calculated from GPC measurements of polymeric precursors aminolysed by 1 -aminopropan-2-01. (column 1.6 X 90 cm, packed with a mixture of Sepharose 4B and Sepharose 6B 1 :I; eluent was 0.05 M Tris (hydroxymethyl) aminomethane buffer containing 0.5 M NaCI, pH 8.0). 50°C acetone HPMA + MA-Gly-Phe-Leu-Gly-ONp

Monomers HPMA

ac~loylglycylphenylalanylleucylglycyl ~-nitrophenyl ester (MA-Gly-Phe-Leu-Gly-ONp) was prepared as follows.

-

P-Gly-Phe-Let&Iv-ONp (Precursors 1 and 2)

azobisisobu~ronitrile,

24 h

Biocompatibility of HPMA copolymer conjugates: B. Rihova et al.

Preparation

of HPMA

copolymer-ADR

conjugates

The amount 2-3

A calculated solution

amount

of ADR

of polymeric

The solution

was

precursor

stirred

amount

of triethylamine

reaction

was allowed

case

of precursor

inactivate isolated

wt) in DMSO. An equivalent

was

then

slowly

added

groups)

below.

hydrochloride

was

added

and the polymer

In the

case

added.

(to was

of precursor

and subsequently

were

and the

in the dark for 3 h. In the

I-aminopropan-2-01

of triethylamine

to a

1 or 2 (15%

any remaining-ONp

galactosamine

was added

at r.t. in the dark.

to continue 1,

as described

amount

hydrochloride

2,

an equivalent

The reaction

mixture

of free ADR

mg of copolymer

extracted

with

was

a mixture

& $i

(I mol-’

= 9800

Inbred

strains

Experiments

were

performed

females

of inbred strains A/J of Physiology,

Prague, Czechoslovakia.

remaining-ONp

groups.

(4: 1). After filtration, and reprecipitated

unbound

Sephadex

ization

The amount photometrically

see Table of bound

using

galactosamine

E;::

content

described”,

4 h, the sugar content

of ”

in methanol

was dissolved

was

Yield

eluent:

was isolated character-

1.

AD8

was determined

= 9800

(I mol-’

polymers

was

spectro-

cm-‘).

The

estimated

. After acid hydrolysis at 100°C

was determined

1. ADR +

P-Gly-Phe-Leu-Gly-ADR

2. Aminopropanol Sample

*

2 2. Galactosamine

1

Gly-Phe-Leu-Gly-ADR

1. ADR Precursor

Two

different

Aminopropanol

During

Academy

experiments,

of Sciences, the mice were

Three

as for

strains

= H -

of a solute

different

and two

of

2b) were

(A/J

(ten mice

routes

different

mice

immunized

2”,

samples

per experimental

(intravenous,

doses

= H -

with

intraperitoneal

of antigens

(300,~g

per immunization)

copolymer

were applied five times in using one of

conjugates

two different

schedules,

either

mice were exsanquinated -70°C

were

used.

or

100 pg of copolymer

Drug-

daily or every third day. The

and the serum taken and stored at

3 or 6 d after the last immunization.

level in serum

was determined

‘Gly-Phe-Leu-Gly-

Detection

The antibody

by ELISA.

2

to microplates

proceeded

aliquots

by ELISA test

was carried out as previously

slovakia)

overnight

were

and

gelatine

rinsed

incubated

and

1%

more rinsings were

bovine

with

filled

(10

mg/ml).

The next day, the

phosphate-buffered

1 h in PBS serum

100,~l

day,

the

microplates

albumin

(BSA).

PBS with

1% BSA.

removed,

rinsed

After

1,2_phenylenediamine The

reaction

was

(5 mg/lO stopped

after

10 min

was

determined

reader

(Minireader

590,

Dynatech)

Isolation

of mouse

Spleens

were

Iscove’s

modified

washed

three times

homogenized

in a tissue

Dulbecco’s

medium.

in an ice-cold

medium

the ethidium

bromide-acridine

orange

of human peripheral

fugationz4, medium estimated

by

20~1

2 M

the ELISAnm.

counted

by the ethidium

in

homogenizer The

cells

density

Iscove’s

in Turk

(5 min. 1 100 was estimated

g) by

solution.

(PBL)

Isolated

from the

gradient

modified

bromide-acridine

Biomatenals

in were

solutionz3.

(PBL) were

Ficoll-Hypaque

suspended and

with

blood lymphocytes

blood lymphocytes

blood

with

splenocytes

The viability

Peripheral

H202

was added.

at 492

in Turk solution.

whole

the conjugate

using

and counted

Isolation

1 :500

were diluted in

ml substrate)

and absorbance MR

horseradish

and 0.015%

H,S04

of the

IgG diluted

1 h of incubation, rinsed

five

20, the

at 4°C. On the

and

porcine antimouse

microplates

After

dilutions

was added. The tested sera and the conjugate was

0.02%

Tween

of different were

saline

containing

tested sera and the plates were kept overnight peroxidase-conjugated

Czecho-

were filled with

PBS and PBS with 0.2% with

Adsorp-

Dalecin,

at 4°C. Wells

with

for

reported14.

(Koh-i-Noor,

of 100 ,ug of antigen

microplates

wells Sample

of antibodies

tion of antigen

next

Figure 1

and

OScSn from the

conditions.

1 or 2 in the form group).

(PBS)

P’

galactosamine 3.

Czechoslovak

inbred

OScSn

Detection 1

12 wk old males

using an amino acid

analyser.

Precursor

standard

on lo-

and C57BL/l

Immunization

or oral)

subsequently

80%;

kept under

C57BL/l

chromatography

fraction

The polymer

by

and the

ether

and diethyl ether

5 X 50cm.

weight

and freeze-dried.

of polymers,

previously

(column

evaporated.

in water

was dissolved of acetone

to gel filtration

LH-20

isolated diethyl

ADR, the polymer

The high molecular

and methanol dissolved

was and

the polymer

and subjected

methanol).

polymer of acetone

into a mixture

(4: 1). To remove in methanol wing

The

into a mixture

of Na2S04

spectrophotometrically

of mice

Institute

precipitation

and

M Na&Os/

) in ethyl acetate.

cm-’

at the

any

(0.2

dried with a small amount of ADR was estimated

at r.t. for 16 h in the dark. 1 -aminopropanto inactivate

as follows:

in 1 ml of water

of 1 ml buffer

concentration

2-01 was

end of this time

estimated

pH 9.8) and 2 ml of ethyl acetate. The organic layer

NaHCO,,

was separated,

was then stirred added

was

dissolved

The

centri-

Dulbecco’s viability

was

orange solutionz3.

1989, Vol 10 July

337

Biocompatibiliiy of HPMA copolymer conjugates: 8. Rihova et at.

[JHj thymidine incorporation by mouse splenocytes or human peripheral lymphocytes

RESULTS

Cells were cultured in Iscove’s modified Dulbecco’s medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS), gentamycin (4Opg/ml) and 5 X 1 OS5 M 2-mercaptoethanol. To estimate cell proliferation, [3H] thymidine incorporation was measured in 96-well microtitre plates (Flow Laboratories, UK) using nonstimulated ceils or cells exposed to Con A (Pharmacia, Uppsala, Sweden, concentration 2 pg/ml in the tissue culture medium). Each well contained 200~1 of cell suspension (5 X 1 O5 mouse splenocytes, or 2.5 X 1 O5 human peripheral lymphocytes) and 50~1 of medium containing Con A and the appropriate HPMA copolymer conjugate. The microtitre plates were incubated at 37°C in a humidified 5% CO, atmosphere, and on the third day 37 kBq (1 +uCi)of [3H] thymidine was added. After 6 h, the cells were collected using an automatized cell harvester, and the amount of incorporated radioactivity was determined in a liquid scintillation counter (Nuclear, Chicago, USA). The results were calculated as the arithmetic mean of the c.p.m of three to four individual wells. The Student’s t-test was used to determine the statistical difference between sample average values.

Antibody response of inbred mice with different genetic backgrounds following administration of ADR-HPMA copolymer conjugates

Colony-forming

unit-spleen

technique

The method of Till and McCulloch25 was used. After exsanquination, the bone marrow was isolated from both femurs of immunized mice and after repeated washing with tissue culture medium (RPM1 1640, Sigma), the concentration of cells was adjusted to 5 x IO5 cells/ml. In all experiments, the viabilitg3 exceeded 95%. The donor bone marrow cell suspension was then injected i.v. (0.2 ml containing 1 x 1 O5 cells) into recipient mice which had been X-irradiated by 6oCo (8 Cy). Mice were killed 8 d after transplantation; their spleens were removed, fixed in Bouin and the number of CFU-s was enumerated. One experimental group consisted of ten recipients which were transplanted with the suspension of cells, pooled from bone marrow of eight mice injected either with free ADR or with HPMA copolymer conjugates.

Tabte 2

Immunogenicity

of HPMA copolymers-ADR

Inbred mice were injected five times with ADR bound to biodegradable HPMA copolymers, without (sample 1) or with (sample 2) targeting galactosamine moiety. For immunization, both samples were applied only as a solution, as only this form might be used for medical treatment. For comparison, two doses i.e. 3OOpg of copolymers (22-25,ug of ADR) or 100,ug of copolymers (7-8~9 of ADR) per immunization were used as it was previously shown9 that 150 pg of ADR given in five consecutive doses is pharmacologically active against mouse leukaemia L 12 10. Serum antibody level was tested on day 3 as well as on day 6 to evaluate the kinetics of the antibody formation. Control groups of animals which were not immunized showed a natural titre of antibodies against the test HPMA copolymer conjugates which varied between 1/16 to l/64, with no detectable differences between A/J and BlO mice (Tab/es 2 and 3). After multiple immunization (five times) of animals with the HPMA copolymer conjugates there was an increase in the antibody production, but this did not exceed one or two dilutions of sera. This means that the immunogenicity of the injected samples was very low. Intravenous, subcutaneous and oral immunization produced similar antibody titres. An indication of the time course of antibody response can be seen if antibody titres from sera taken on the 3rd and 6th day after the last injection are compared (Table 3). However, at all times, any increase in the antibody level was very low. Neither the dose of the antigen (300~9 or 1 OOpg per immunization) nor use of sample ‘I or 2, influenced the final antibody titre. At the beginning of the experiment, we have assumed that daily immunization, in contrast to the immunization every 3rd day, might lead to some kind of immune tolerance. However, no such results were obtained and both immunization schedules produced similar, very low levels of antibodies (Tab/es 2 and 3).

samptes following daily immunization

Sample

Dose Copolymer

Route of application ADR

Antibody titre A/J mice

C57BVl

OScSn mice

Cg) P-Gly-Phe-Leu-Gly-ADR

,

Gly-Phe-Leo-Gly-ADR

300

25.5

i.v. s.c. orally

l/128 l/128 l/128

l/l 28 l/256 l/512

100

8.5

iv. S.C. orally

t/128 l/64 t/1 28

l/512 l/512 l/256

iv. S.C. orally

t/256 f/128 l/64

l/l28 f/256 l/32

iv. s.c. orally

t/64 l/32 T/64

l/128 t/64 l/32

t/32

l/64

300

2t.9

‘P ‘Gly-Phe-Leu-Gly-gal

100

None (control)

7.3

-

Ten mice pergroupwere immunized daily(fivetimes). Six dafterthe lasttreatment,the an average of ten individually tested sera. ‘P = HPMA copolymer.

338

Biomaterials

1989. Vat 10 July

micewereexsanquinatedand

theserastored

at -70°C.

Numbers represent

Biocompatibility

Table 3

lmmunogenicity

of HPMA

copolymer-ADR

Sample

samples

following

immunization

Dose Copolymer

every

third

, Glv-Phe-Leu-Glv-ADR

300

21.9

100

7.3

orally

l/16 l/l 28 l/64

l/16 l/l 28 l/128

l/64 l/128 l/32

l/256 l/256 l/64

I.“. SC. orally

l/256 l/256 l/l 28

l/256 l/256 l/256

l/l 28 l/64 l/64

l/l 28 l/128 l/l 28

iv.

l/64 l/64 l/32

l/128 l/64 l/32

l/32 l/32 l/64

l/69 l/69 l/69

l/32

l/32

l/16

l/16

S.C.

orally None

OScSn mice 6th day l/128 l/128 l/l 28

I.“.

Glv-Phe-Leu-Glv-gal

C57BL/l 3rd dav l/32 l/128 l/64

S.C.

*p \

et al.

l/128 l/64 l/128

orally 8.5

B. Rihova

l/64 l/64 l/64

i.v. S.C.

100

A/J mice 6th day

3rd day 25.5

conjugates:

Antibody titre

ADR

300

copolymer

day

Route of application

lug) P-Gly-Phe-Leu-Glv-ADR

of HPMA

(control)

Ten mice per group were immunized every 3rd day (five times). On the 3rd and 6th days after the last treatment the mice were exsanqumated and the sera stored at -70°C. Numbers represent an average of ten individually tested sera. *P = HPMA copolymer.

Table 4 HPMA

Spleen

colony-forming

copolymers

containing

units

(CFU-s)

detected

in irradiated

recipient

mice

afrerinjection

of bone

marrow

harvested

from mice qected

wth

free ADR

Sample

Immunization protocol

Route of appllcatlon

Number of CFU-s + SE per spleen detected on day 3rd

P-Glv-Phe-Leu-Gly-ADR

6th

I.“. A

26 + 4 29 + 3 30 t 4

S.C.

orally 26 + 5 25 + 3 27 * 2

I.“.

B

S.C. orallv

A

S.C.

27 + 4 28 + 4 27 + 5 30 + 3 281 5 30 + 5

I.“.

,

or

ADR

Glv-Phe-Leu-Glv-ADR

orally

*p \ Glv-Phe-Leo-Glv-gal

i.v. B

23 t 3 26 + 1 27 + 4

SC.

orally

29 + 5 27 + 3 27 + 3

I.“. ADR

A

1412 18- 3 28 + 5

S.C.

orally I.“. B

5 + 1 11 t3 25 + 5

S.C.

orally

13 f 3 19 1 3 27 i 5

None (control)

25 + 4

A = Immunization daily five times with 300~9 of polymer (22-25,ug of ADR per immunlzatlon). On the 6th day after the last treatment cells from bone marrow were isolated. B = Immunization even/ 3rd day. five times with 300pg of polymer (22-25~9 of ADR per immunization). On the 3rd and 6th days after the last treatment cells from bone marrow were isolated. Data are expressed as the average of the triplicate f SE. *P = HPMA copolymer.

Toxicity of free ADR and ADR bound to HPMA copolymers against haematopoietic precursor cells in bone marrow of inbred mice

venously

precursors

was

shown

to be toxic

in bone marrow

only if injected

subcutaneously.

After

significant

on the number

spleens

effect

of recipient

oral mice

against

administration, (Table

of CFU-s 4).

ADR

haematopoietic intravenously there

was

detected injected

about

bone marrow Three

Free ADR

as a total dose of 125 1-19 in five separate

eliminated days

80%

of haematopoietic

stem

taken on the 3rd day after the last treatment. later,

the

number

of CFU-s

measured

spleen of recipient animals was in comparison or

by 50%.

no

function

in the intra-

which

suggests

after ADR

Following copolymer

doses

cells from

rapid

treatment

recovery

in the

only decreased

of bone

marrow

(Table 4).

administration

carrier with or without

of ADR

bound

galactosamine,

Biomatenals

1989,

Vol

to a HPMA there was

10 July

339

Biocompatibility of HPMA copolymer conjugates: B. Rihova et al.

no detectable toxic effect on haematopoietic precursors in bone marrow. The minimal decrease in the number of CFU-s seen in the recipient mice when bone marrow was taken on the 3rd day after intravenous injection of drug-copolymer conjugate with galactosamine (Table 4) was no longervisible on the 6th day after treatment.

Table 6 Inhibition of [3H] thymidine incorporation into human peripheral lymphocytes by free ADR or ADR bound to HPMA copolymers Sample

Copolymer

Table 5 Inhibition of [3H] thymidine incorporation into mouse splenocytes by free ADR or ADR bound to HPMA copolymers Sample

Concentration Copolymer

ADR

[3H] thymidine incorporation (in % of control)

(pg/mU *P-Gly-Phe-Leu-Gly-ADR

2000.0 200.0 100.0 50.0 20.0 2.0 1 .o 0.2

170.0 17.0 8.5 4.25 1.7 0.17 0.085 0.017

,Gly-Phe-Leu-Gly-ADR

2000.0 200.0 100.0 50.0 20.0 2.0 1 .o 0.2

146.0 14.6 7.3 3.65 1.46 0.14 0.07 0.014

17.0 30.0 41.0 73.0 95.0 100.0 100.0 100.0

200.0 20.0 10.0 2.0 0.2 0.1 0.02 0.002

2.0 3.0 3.0 4.0 23.0 39.0 69.0 91.0

p\

Gly-Phe-Leu-Gly-gal

ADR

1 .o 5.0 12.0 61.0 92.0 91.0 93.0 94.0

Each well CGntamed 37 k8q of 13H] thymidine and 2 pg/ml of Con A. Controls (5 “: 1 O5 calls/well) = 400.000 c.p.m/well = 100% of proliferation. Data represent the mean of the triplicate. *P = H PM 4 copolymer.

340

Biomaterials

1989, Vol 10 July

ADA

[3H] thymidine incorporation (in % of control)

(fig/ml) *P-Gly-Phe-Leu-Gly-ADR

500.0 50.0 25.0 5.0 0.5 0.05

41.5 4.15 2.07 0.41 0.041 0.004

15.0 51.0 62.0 88.0 100.0 100.0

,Gly-Phe-Leu-Gly-ADR

500.0 50.0 25.0 5.0 0.5 0.05

36.5 3.65 1.82 0.36 0.036 0.004

6.0 42.0 51.0 78.0 100.0 100.0

The effect of free ADR or ADR-HPMA copolymers on [3H] thymidine incorporation by human PBL and mouse splenocytes in vitro Proliferation of mouse splenocytes and human PBL was measured in the presence or absence of Con A. The reason for experiments without Con A was to determine if copolymer-bound ADR, as a potentially foreign substance, does not even stimulate proliferation of mouse splenocytes or human lymphocytes if given in very low non-toxic concentrations. No such results were obtained (so the table is not included) and only an inhibitory effect of free ADR as well as copolymer-bound ADR was detected (Tab/es 5 and 6). Therefore, these materials are not themselves mitogenic. Free ADR was a very potent inactivator of Con A induced cell proliferation; concentrations as low as 0.02 pg/ml decreased [3H] thymidine incorporation by mouse splenocytes by 30%. Human PBLs are even more sensitive to free drug. A concentration of 0.005 pg/ml was sufficient to induce suppression in human lymphocytes equivalent to that induced by O.O2pg/ml ADR in mouse splenocytes (Tab/es 5 and 6). ADR bound to HPMA copolymers showed much less ability to inhibit [3H] thymidine incorporation in both cell types (Tab/es 5 and 6), 2 pg/ml of both polymer-bound ADR samples induced 40 and 50% inhibition of human PBL

Concentration

p\

Gly-Phe-Leu-Gly-gal

ADR

2.5 0.5 0.05 0.005 0.001 0.0005

2.0 4.0 14.0 64.0 85.0 100.0

Each well contained 37 kBq of [3H] thymidine and 2,ug/ml of Con A Controls (2.5 x 1 O5 cells/well) = 20 500 c.p.m./well = 100% of proliferaton. Data represent the mean of the triplicate. *P = HPMA copolymer.

proliferation and 4pg/ml was necessary to inhibit to the same degree proliferation of mouse splenocytes. There was no marked differences between HPMA-copolymer ADR samples with or without galactosamine.

DISCUSSION If HPMA copolymers are to be used therapeutically, their biocompatibility must be examined carefully. Previous experiments have studied various aspects of polymer biocompatibility. The rate of enzyme degradation of the oligopeptide side chains in HPMA copolymers has been well documented2,4,6. Incubation of HPMA homopolymer, and copolymer with P388D cells in vitroz6 showed no loss of cell viability or lactate dehydrogenase release with concentrations up to 1 mg/ml. HPMA copolymers were found not to accumulate specifically in any tissue after administration in viva if their molecular weight is reasonably low. Seymour et al.” have shown that lz5 I-labelled HPMA copolymers of average molecular weight below 45 kD are quickly eliminated from rats by urinary excretion. After intravenous application, blood clearance was found to be molecular weight dependent, 50% clearance taking 3-72 min depending on the molecular weight average of the sample tested. Tissue or cell accumulation of HPMA copolymers, either at the site of the injection, or in spleen, liver or wall of the small intestine was seen only with the copolymers of very high molecular weight average (778 kD). Considerable attention has already been devoted to testing the immunogenicityof the HPMAcopolymers’4-‘6. It was found that HPMA copolymers do not only fail to induce an immune response against themselves, but they even have the capacity to decrease the antibody response against immunoglobulins or other proteins bound to them as targeting residues (Flanagan et a/., unpublished results). Experiments testing the interaction of HPMA copolymers with a complement showed that they only activate the

Eiocompatibility

complement at very high concentrations (20 mg/ml) which would never be used therapeutically. Lower concentrations

toxicity

in

(2 mg or 0.2

marrow

stem

alternative

mg/ml)

did not activate either the classic or the

complement

activation

to HPMA

residue

copolymers

galactosamine,

production

did not cause

attributable

drug which, eventually

even if bound penetrate

subsequently However,

inhibit

stantially

change

administered

ability

antibody

jugates

containing

ADR

which (Rihova,

ADR were

lower

indicates was

compared

the low

after

orally”.

comparison

bovine

con-

gamma

tract

However,

and oral

is metabolized be given

are attempts

reactions

different

and the routes

drug

the drug at the target

delivery

is not only to

For this reason, we have in

the

of free

past,

compared

the

toxicity

1.2 and anti-la

antibodies

as targeting

haematopoietic

moreover, a damage

of

hepatotoxicity of

the

myocardium

against

comparing

HPMA

free ADR marrow.

-80%

when

of ADR

The drug-copolymer

No

carrier,

non-toxic,

containing

galactosamine against

bone

intravenous

administration.

The anthracycline normal

via membrane

were

mitosis,

interaction

and

bound

human

conjugates

1.2

antibodies

be concluded

any

of the

against

bythe

undesirable

drug

immune

to the

haematopoietic

that

recipient

copolymer

precursors

of proliferation

in

of normal

decreased.

ACKNOWLEDGEMENTS The

authors

wish

Semoradova

to thank

for excellent

Mrs

D. Plocova

technical

and

Mrs

H.

assistance.

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toxicity

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of

obtained

cells

eliminated

without

antibiotics

by intercalation

not

lymphocytes

and,

signs

of polymeric

exerted

cytotoxicity

the

do

it can

as well as inhibition

most

by enumeration

stem

cytotoxicity

whereas

nor

if daunomycin-

anti-Thy

are well tolerated

bone marrow

in bone marrow

results

cells were

in the form

completely

preventing

marrow

of free ADR and ADR

conjugate

samples

of [3H] thymidine

T-cells

presented,

By attachment

L. lllum

days after the last treatment

No such profound

administration

inhibition

spleen

prodrugs

and

Release

conjugates

measured

on haematopoietic of these

data

reactions.

the

cells in liver and

precursors

very similar

(Table 4). Three

between

splenocytes

targeting

organism

the

observed.

copolymer

the effect

copolymers

marrow

mainly

were

reduced

In this study,

the

polymeric

against

in bone

of Kupffer’s

haematopoietic

was considerably of CFU-s’.

From

and heart toxicity was seen after the application

daunomycin-HPMA

toxicity

when

irritation

with

a

(Tables 5 and 6). This is due

mouse

mouse

copolymer

and against

pg), it eliminates

precursors

a significant

and

is highly toxic; when given

(total dose of 1 50-600

rntravenously

showed

both cell types,

was tested33.

anti-Thy

residues,

stem cells in bone marrow

liver and heart7. Freedaunomycin of the

daunomycin

bound to an HPMAcopolymer-bearing

haematopoietic

copolymers

differences

a significant

into

to ADR

(Tables 5 and 6). Unlike

site, but also to eliminate

its toxic effects on normal tissues. daunomycrn

neither

lymphocytesin

more sensitive

of

importance.

The aim of the targeted

as HPMA

incorporation

to the

to use HPMA

delivery2a

after

com-

as well

conjugates

intraperitoneal that ADR

and so cannot

there

which

of the tested

for gastrointestinal

of defence

than those of

globulin’5,

copolymer

it is known

the

were

on

compared

are targets for these drug-copolymer

as we have detected

HPMA

containing

ADR

bone

have

activity against

galactosamine

HPMA

we

to HPMA

no significant

that

the effect

splenocytes

and there were fact

and

To investigate

and human peripheral

bound

et al.

and dose-limiting

on [3H] thymidine

lower inhibitory

lymphocytes

B. Rihova

cardiomyopathy

of free

with or without

production

On average,

ADR

considerably

to the

specificity

lymphocytes

than mouse

incorporation

previous

copolymers

of HPMA

is of a great

accumulate

simul-

unpublished

with

results).

intravenous,

conjugates

treatment

not sub-

copolymer

accord

immunogenicity

in the gastrointestinal patient

in

against HPMA

even when

copolymer

HPMA

against

(Rihova,

little or no antibody

lmmunogenicity

application,

did

by four orders of magnitude

a very

pounds.

results)

of HPMA

unpublished

against

of

with HPMAcopolymer-daunomycin

titres of the antibodies antibodies

antibodies.

injection

response

is

show

immunization

conjugates

produce

SRBC antigen

Poor immunogenicity

might

cells and

to the dose and immu-

in our

results).

observations

to

as an

activity

by mouse splenocytes

the free form,

carrier,

conjugates:

lymphocytes,

conjugates

vitro. The human

to the copolymer

(according

the

suppressive

treatment

described

taneously

following

antibody

limited includes

of normal

copolymer-ADR

(Tables 2 and 3). This could

case,

ADR

schedule

significant

the

copolymer

cell suppression.

to the fact that ADR is a cytotoxic

their the

of

the targeting

into the immunocompetent

it is not

copolymer-bound

that the attachment

in addition,

against the conjugate

be theoretically

nization

and

display

humans

proliferation

pathway17.

In this paper, we have shown ADR

All processes

of HPMA

B. and

P., Strohalm,

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agents

copolymers. L1210

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33