Symposium on ‘liver selective fibrosuppression’

Panel discussion

Chairmen: Professor Dr. E.G. Hahn, Erlangen. F.R.G.

Dr. V. Giiozler, Frankfurt, F.R.G.

E.G. Hahn: Is there anybody’wbo vooid like io ask a

question to Dr. Pihlajaniemi about the role of prolyl4.

hydroxylase in collagen formation? I think it was an ex-

cellent overview, and I was surprised how many molec-

ular data were available, but my impression was that

the interpretation of most of these data still is in a hud-

ding phase. Is that correct?

T. Pihlopniemi: We certainly still have a lot to do. For in.

stance to study the regulation of prolyl 4-hydroxylase

synthesis. That is something, that we are trying to

address. For examole. with this F9 cell svstem that I

mentioned. And also we would like to produce large

amounts of the fi subunit and also of the prolyl 4-hy-

droxylase tetramer, in order to be able to crystallize

both enzymes and study the structure better than it has

been done.

Question: You told US that the enzyme is locaicd in the

rough endoplasmic reticulum membrane and is active

there. You also pointed out that there is an activity di-

rected to disultidc link formation. Is it known whether

this activity is residing in the endoplasmic reticulum

membrane?

T. Pihlojonicmi. Protyl4-hydroxylare is only very loosely

bound to the membrane of the endoplasmic reticulum.

The site of prolyl 4-hydrorylase is definitely the endo-

plasmic reticulum only. The protein disulfide isome-

IBEB, the 6 subunit of prolyl 4.hydroxylase is also only

found in tbc endoplasmic reticulum.

Question: Of course this is impor<znt tar the question

whether the capability of formmg disulfide crosslinks

ih:ir anything to do with collagen formation. Is that

known at all?

T. Piklojaniemi: I cannot answer your qertion. We

know that in evolutionary lower forms, e.g., Chlamy.

domonas, prolyl4-hydroxylase consists of one subunit.

That enzyme does not have any potent protein disulfide

isomerase (Pal) activity, The! ssggas tb.? potent

PDI activity is not needed for prolyl4-hydroxylase ac-

tivity. We have expressed mutant PDI that does not

have any I’D1 activity. Next we will express bath the a

subunit and the/3 subunit in the same bacteria and these

bacteria will prcwmebly form prolyl4-hydroxylase te-

tramer. WC will assay whether these enzyme tetramers

hax prolyl4-hydroxylase activity. Thus we will deter-

mine the effect of the PDI mutations on the hydroxylat-

ing enzyme activity.

Question: Do you also inhibit the PDI active site by in-

hibition of the prolyl4-hydroxylase activity?

H.M. Hnnowke-Abel: We did studies with ‘2.oxoglota-

rate antagonists. What you have to look at is a test sys-

tem where an inhibitor is used that inactivates the 01

subunit. and then PDI activity has to be assayed. We

did not do these tests.

Question: Do you know whether the mRNA, for instance

for prolyl Chydroxylase. would increase or change in

any way?

V. Giinzler: We have tested competitive and irreversible

inhibitors of prolyl4-hydroxylase in fibroblasts and we

always observed a drop in the message of the collagen

types 1 and III. I do not know whether this is a universal

phenomenon but 1 think Dr. Cl6ment has observed a

simi!ar effect with HOE 077 in his cell cultures. There

was also a slight decrease of the a subunit and B subunit

message, but it MS not significant.

Qsewinn. Do you know anything about the nalural sub-

strate for the PDI subunit of prolyl4-hydroxylase?

ic suds. It IS part of the xenobiotiL metaholiiing func-

tion of the !iver to polarize agents. And cnce they are

excreted they cannot reenter cells. unless they are in-

completely polarized and they enter a hepatic recircu-

lation mechanism. Dr. Volz presented data on how fast

the compound is excreted. After 1 h, the compound is

dlstnbuted all over the rat with preference to the portal

system end the liver. After 4 h, the compound is found

only in the got and the winary system. Reentry of a po-

lar agent into other tissues once it has been partially or

totally metabolized by the liverisvery unlikely.

Question: Is there any evidence that the active netabo-

life M2 is able to cross the endoplasmic reticulum mem-

brane?

H.M. Hanauske-Abel: When we did the tests ,?ith th: pu-

rified enzyme and with the enzyme mated by its micro-

somal membrane we did not have agents that were

blocked at one carbnnyl group only. Thus we can spec-

ulate whether an ester in position 4 still inhibits the en-

zyme in any of these test systems. The phammcokinet-

its definiteiy have to be worked our and then one ha to

go back and test the identified agents that are produced

by the liver in any one of these systems. At this time

one can say that there appears to be a highly specific

cjtdke mechanism in the membrane of the endoplas-

mic reticulum that preferc 2,4-PDCA. ‘tie do not even know whether it is an active uptake. because we did not

have the radioactive 2,4_PDCA when we did these

studies. We on!y could measure the enzyme activity in-

side the microsomes as an indicator of whether the

compound is there. Now when we have !!w radiowtive

compound we can measure the uptake.

V. Gfinrler: We must not make the mistake to compare

the effects of the esters in fihroblasts to those in the

liver. In fibroblasts, the proinhibitorlinhibitor conver-

sico is ZPPo:eot!jj daoe by cytoro!i: i::;;;ies and the

active compound most go through the endoplasmic re-

ticulum membrane. In the liver. a lot of metabolizing

enzymes are localized inside the cisternae of the endo-

plasmic reticulum. It may very well be that an active

metabolite in the liver is generated in the endoplasmic

reticulum and need not cross any mombrae before act-

ing.

Quesrion: We all agree that mesenchymal cells like lto-

cells or madtfied Ro-ceiis are the malor collagen pro-

ducers in the liver. Do you have any hypothesis or is

there any precedent of the cell to cell transport of acti-

vated metabolites from the hepatocyte to the Ito-cell?

‘T. Pihlajwdemi: The enzyma activity can be assayed in

vitro by ribonocleaae A activation. PDI activity is proh-

ably important for disulfide-bound formation of many

secreted proteins.

Question: Do you alw mean the subunit which is present

in the prolyl4-hydroxylase?

T. Pihlajnniemi: That subunit is the same enzyme which

exists as a free protein in the endoplaxnic reticulum.

Thus PDI is b&wed to be the in viva catalyst of di-

sulfide bond formation of many secrered proteins. The

function of PDI activity in the prolyl 4-hydroxylase te-

tramer is not known.

Question: Maybe it is not only this designed dmg that in-

hibits, but alsorome metabolites?

R M. Hanauske-Abel: Absolutely. I think it is very clear

form the presentations that we have a new agent which

does something positive in the animal system. Howev-

er, every new approach presents more questions. be-

CBIJS~ the systems are so complex. I am not at ali con-

cerned about all the white spots on the map. I am very

hopeful that we fill them with rime. So 1 would not he

surprised if the compamd. the design of which was

based on orbital interactions, turned out not to be the

main inhibitor. It does not need a free 4.carbmyl moie-

ty to inhibit prolyl 4.hydroxyiase. At ibe timr we did

these studies we did not have agents that had one car-

bayI group free and the other carboxyl group blocked. Thus we could not go through the purified enzyme, the microsomal enzyme, the cellular enzyme, and the tis- sue system to look at the effects of these monoe~ten or monoamides. But the key point is that without any screening, we came up with B very promising agent.

Now one has to fill in the blanks.

Quesfion: How do these compounds or metabol;;es go into the cell and how might they get out? Do you have

any suggestions or any information as to whether such

polar compounds could get, for instance, into fat-stor-

ing cells by pinocytosis or in other ways than the usual

penetration of the membrane?

H.M. Hounausk--AI r!: I do not yet have any exoexi-

mental data, but it is always helpful to have a hyp:.the-

sis that directs your experimental approach an6 this

was what we had here. As soan as you have a polar

agent that is generated inside of the cell it has a hard tirw getting out. It needs a special transpat system.

The liver is known to have transport systems for organ-

S68 PANEL DISCUSSION

sis of any etmfogv is that we prevent the destruction of

the normal functional architecture of the acinua which

is crucial for the function of the liver. All the evidence

that we presently have is that if you prevent fibrosis and

destruction of the acinus by suppression of either in-

flammation or fibrogenesis you will prevent cirrhosis

and its complications. This is true for chronic liver dis-

ease. II may be completely different for acute liver dis-

ease and Ccl, intoxication of tats is an acute liver dis-

ease. It is acute necrosis with some chronic sequefae

but they stop immediately if you stop Ccl+ It is not

comparable to any of the known disease entities that we

describe as liver fibrosis or liver cirrhosis in man.

Question: Do the livers actually shrink? We see very little

fibrosis in the drug-treated animals compared to the an-

imals not treated with the drug. CCI, toxicity is really

damaging the cells. Then the cells die and are replaced

by fibrous tissue. So if you stop scar formation the liver

should then simply become smaller.

M. &c&e!: On an awa- a* relative weight of the livers _- . .._

is not changed in &her U&treated or drug-treated

animals. There iare some animals which have a de-

creased liver weight of 3-4 g in the CCl&eated groups. But these animals are in a very bad condition at

autopsy, that means these are final stages of cirrhosis. Normally you do not find those conditions in drug-

treated animals.

Commenr, W. Horn: You would not even expect that,

since regeneration of hepatocytes should occur in drug-

treated animals. Imagine a cirrhotic nodule, necrosis

within this nodule, and the cells trying to regenerate. If

you prevent formation of the ffbrous capsule and re-

placement of necrotic material by collagen, cells will

have space to regenerate.

Question: Have you tried other models of fibrosis?

M. Bickel: We have tried a couple of models. Firstly, we

coadministered ethanol end Ccl, in rats as has been

published by several groups. We could not reproduce

these rerufts. We got a fibrosis independently of wheth-

er we aoded ethanol to CCI, or not. Secondly, we ~4

tbe model with a choline-deficient diet. We treated the

enimalr. for 6 months, but they did not develop fibrosis.

TXrdly, we edministered porcine serum to rats, but

again we could not produce fibrosis after 6 months of

treatment. The bile duct ligation model seems to be one

of the best models for producing fibrosis il tfv rat.

H.M. Hanaurke-Abel: No, I do not know anything about

transfer from a cell that partially activates the prodrug

to a neighbouring cell where the compound exerts its

presumed effect. If you have a cell thai is able to acti-

vate a compound and a neighbouring cell that is not

able to activate the compound you stiff may have pro-

found disruption of matrix deposition. Because under-

hydroxyfated material may leak out of the cells and in-

hibit the procoftagenlcoffagen conversion. There are

multiple ways to suppress matrix fonnatbn without

transfer of agents from one cell to another.

Question: In your histological pictures from your CC4 rat

model you show that there is a big difference in terms of

septa between treated and untreated rats. Thus, I es-

sume that you have also a reduction of mesencbymal

cells in your treated rats, because there are no septa.

M. Eickel: There are small and !iny septa in the region of

the central vein. Of cane there are other pictures

where the development of septa is clearer than in the

example I have shown. There is a development of septa

even under drug treatment.

Commenl: And therefore the cell population of mesen-

cityymal cells in both septa is similar, because otherwise

you should assume that this molecule is able to inhibit

probferation of mesenchymaf cells.

Question: Is the liver architecture in your drug-treated

animals normal?

M. Bickel: We hzve no measure for normal architecture

of tbc f:vcr, I would not be aware of how to measwe

this or est~m.& it. When you !ook at the sections of the

liver you find that in CCf,treated animals you have af-

ways large and small regenerating nodules separated

by large connective tissue septa. In the drug-treated an-

imals you never see small nodules. There are tiny septa

and the periportaf fields are not destroyed which is af-

ways the cese in the CC&-treated animals.

Cornmen!. E.G. Flc+, Yxr ,-;,;i,.‘ L II vzty critical

one and I may perhaps cite our old mentor ilans Pop-

per who bas alw.vs said, ‘If you can stop fibrogenesis or

could get rid of the fibrotic matrix in liver cirrhosis

you will CUIC chronic liver disease’ Tbf. question still

needs to be challenged. Patients who eventually die

from bver failure or complications of cirrhosis go

through a fibrotic stage. The destruction of the acinus

particularly in alcoholic disease leads to regenerative

nodule fOmx+tion. What we do when we prevent fibro-

S69

Quess~~on: Ic any?hing known aboor the half-life of intra-

cclhdar prolyl 4hydroxylare. of it is irreversibly

blocked?

T. Pihlajoniem~ The half-life of in*raceilular prolyl 4.hy-

droxylase ii around LO h.

Questions What was the result wirh respect to hy-

droxyproline synthesis when you treated your cultured

hepatocytes with the drug?

R CIPmenr- We incubated the hepatocytes wim [“C]pro-

line for 16 h. Tbcn we extracted the acetic acid soluble material from both media and the cell layer and we ob-

served a dramatic decrease of hydroxyproline content m drug-treated cuhores.

Question: How did those doses yen uacd compare wth

the likely blood levels which are achieved in viva?

8. Cl&wnl it 1s very difficult to compare the in viva with

them vitro experiment. We have incubated the cell lay-

erwith4.5mgimlofHOE077.

Quanon: Very high indeed. We are talking about blood

levels of&ml rather than mg/ml.

B. CUmenr: Hepatocytes in culture exhibit rapid phe- notypic changes. They are not actually the same as ic

wvo and they dedifferentiate very quickly. In terms of

fonction we know that only 5% of the tranwiption of

spenfic genes IS main!wed ef!er 24 0.

Que.&n: You showed that collagen synthests was re.

duccd in your hepatocytc cultures and in the co-cul-

tares, but the collagen in the extracellular matrix ap-

peared not to be effected. Is that true?

B. C/&nenr: There is definitely no deposition of collagen

outside of the heparocyte in ~d~???,~ ce!torcs. There is

only deposition of laminin. fibronectin and proteogly-

cans. There is no deposition of collagen around the cell. In ,,o-cultures, there is deposition of colla&en be-

tween the two cell types. We have tested the effect of :IOE j, i ;n this ;y::sm bvt for technical reasans we

had to add me compound only 24 h after me co-culture sysicm i1.d bsso jet op. That means that at this time

there 1s already plenty of matrix deposition between

both cell types. We observed strong inhibition of the in-

tracellular sti ;ning for types ill and IV collagen but no

inbtbition oi rt e e.rtracellular deposition. The strategy

Commenr. E.G. Hahn: This model is very clox to me hu- man situation where you have obstructive Ibile duct dis-

ease, although we cannot compare the molec,dar

events exactly. There is no ideal model for alcoholic

liver disease or chronic active hepatitis in man.

QuesGon: There may be a collapse of the pxenchymal

mass under drug treatment in CClhinduced cirrhosis

Is it not possible that a certain amount of collagen or

connective tissue is necessary for an ordered regenera-

tion? The therapeutic range for these agents might be

OBTTOW. They should not completely repress col!agen

biosynthesis.

M. Bickel: We never found an average antifibrotic effect

of more than S&60% with HOE 077. This excludes a

complete disappearance of collagen from the liver.

There will always be enough collageq present as a ma-

trix for an ordered regeneration of hepatocytes.

Question: If the half-life of HOE 077i is only 2 h, and 1 as-

some you gave the drug only once a day. do yoo think

this is an appropriate method for really Icaking at a bio-

logical process such as fibrogenesis?

M. Bickel: We always gave the drug twice daily

Question: Wdh a half-life of 2 h, the concentration is low

after 8 h.

M. Bickel: But we do not know the concentration of the

inhibitor that is necessary to inhibit the prolyl4-hydrox-

ylase within the endoplasmic reticulum. Measuring

concentrations in urine and serum lust gives us a hint

that there is enoogb substance available. We have done

experiments with ones daily dosing and there was a

marked decrease of the antifibrotic effect. Compounds

similar to HOE 077 were administered subcutaneously

by osmotic minipumps that gave steady state delivery

over 24 h. Even under these conditions we have oat

been able to show a more pronounced antifibrotic ef-

fect. Twice daily edministration of the drugs is suffi-

cient.

Qzesrion: Dr Volz. would you comment on how long

yea can spe ar?i+ .n cells in your total body auto:a-

diographies?

M. Voh: After 4 h the concentration of HOE 077 in the

liver is decreased to l/IO of the concentrations 1 h after

dosing. We can estimate the half-life in the liver to be

1-2 h.

s70 PANEL DISCUSSION

producible effect in animal models and we are trying right now to understand how this effect is produced on

the cellular level, and by which metaholites of HOE

077. This compound is a pmdmg, which means that it is

not an inhibitor of prolyl4-hydroxylase itself. It seems

to be activated only in the liver. Therefore, a general-

ized connective tissue damage, e.g., damage to the ves-

sel walls to the canilage, or to the growing organism

would be avoided.

The limitation of this approach is a reduced activa-

tion of the drag in patients with strongly reduced liver

function. This may mean that in very late stages of the

disease the therapeutic effect may decrease. But I think

the experiments tell us that it should be possible to im-

prove the efficacy of future compounds.

Thank you to all the speakers and to our auditorium.

now is to preiocubate the cell with HOE 077 and then

to co-cultivate both cell types.

Question: What was the dose giveo to the vc!unteers h

the phase I study?

W. Horn: Up to 1500 mg per person. This corresponds to

the effective antifibrotic dose in rats.

Concluding remark

V. Glinzlec I think the general impression was, that a lot

of questions had to remain unanswered. And the

reason for this is of course that we are developing a

completely new drug. In doing so, we are entering a

whole new continent of experiments. We do see a re-