CA1312064C - Preparation for haemoperfusion or haemoadsorption - Google Patents

Abstract

ABSTRACT

The invention provides for a preparation for haemoperfusion or haemo-adsorption which consists of silica gel.

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Description

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Prepsration for h~emoper~usion or h~emoad~tDrption The invention relate~ to e prspar~tion for haemoperfusion or haemoadsorption, By haemoperfusion or h~emoadsorption is meant ths adsorptive bonding of ~ubstances dissolved in th0 blood to solid~ which have a large ~urfaca area. 6y this proceae it i8 possiole to r~move exogenic and sndogenic toxins9 such c~ for ex~mple ~mtriptylin, q~inineHCl, dig~xin, digitoxin end methsqualcn, diractly from the blood During the flow-throu3h9 ths conGentr~tion of the ~ub~t~nce whi¢h i~ to be rsmoved9 the ~dsorbate~
drops pr~cti~ally to nil if thare i8 suf~icient ~olid capac$ty and idaal bonding condition~ between the adsorbate and adsorb~nt. With known proces~es, activated csrbon ~nd various ion exchanger3 ar~ used as the adsorbent for haemoperfusiDn. (E~ Wet~els, A. Clombi, P. Dittrich, H. J. Gurland, M. Kessel ~nd H. Klinkmann, "Haemodialysis, Peritoneal dialysis, ~smbrans plasmapheresi~ and relsted proce~esn, Springar-Verlag, aerlin 1986, Pages 66-69 anD 606-608; and H.E. ~r~nz (Hrsg.) "Blood cleansing process"; G. Thiems Verlag, Stuttg~rt 1985, pege~ 15 ~nd 473-477~.

The ~dsorbsnts usad up until now are unsatisfactory in meny rsspects.
Thera ia therefore a nasd for new preparations for haemoparfusion or haemoscl~orp tion .

The object of the invention i8 to provide new preparations for ths ~forementioneo purposes.

1.

~3:~2~4 It has surprisingly been shown that silica gel is an excellent widely available preparation for haemoperfusion or haemoadsorption. Colloidal dense or spongy, formed or unformed silicic having a ~ine pore structure and thus high absorption capacity is generally described as silica gel or siliceous gel.
The sole figure is a diagrammatic illustra-tion o~ an experimental set-upO
A reverse~-phase siliceous gel has proved particularly suitable as silica gel. In the reversed-phase the silica gel contains octylor octadecyl-functional groups covalently bonded to the surface hydroxyl groups. The surEace is thereby substantially non~polar and hydrophobic. Reversed-phase silica gel and its properties are described in the ~ollowing literature R.C. Dunhy, "A dictionary of chromatography" Macmillan reference books, Page 160; C. Horvath, "Reversed-p'nase chromatography", Trends in analytical chemistry, Reference edition 1.1981~ pages 7-8 and K~Ko Unger, "Porons silica", Journal of Chromatography Library, Vol 16, ~lservier Sc. Publ. CoO, AmsterdaM, pages 216-217. A reversed-phase silica gel having a pore diameter of about 10 nm with a particle size of ~0-60 micrometers has proved particularly suitable. This silica gel is subs-tantially non-polar and hydrophobic. The pH value of the aqueous suspension is 7.
When preparing reversed-phase siliceous ~el silica is used which has an average pore diameter of 6-10 nm.
The non-polar dimethyloctylligand was anchored on the surface hy a silaxon bridge whereby the surface becomes hydrobic.
The sur~ace is thereby not completely covered. Some polar silanol groups still exist. Subse~uent treatment with tetramethyl chloric acid reduces the remaining polar silanol concentration. However a true hydrophobic sur~ace is never reached, non even if octadec~l-functional groups are used.

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The adsorbent ~s2d in the experiments is Lichroprap RP-18 ~ (~erck Co.3 Its phy6ical properties are given in TabO 1~ Lichroprep RP-18 i8 a reversed-ph~se siliceous gal.

Tuble 1: Chamicel physicsl dat~ for Lichroprsp RP-15 Pore diamater 10 nm Partlcle size 40 - 60 micrometer Polar/Non-pol~r Non-polar Hydrophilir/Hydrophobic Hydrophobic pH of the ~quaous 3uspen~ion 7 When proparing th~ rev2rseri-pha~e silicaous gal silicQ i9 used which has an ~vera0~ pore diameter of 6- 10 nm, wh~rain the ~ a i8 first cleon0d with acid and than neutrslized by washing out with distilled water. The silica is then drisd overnight ~t 180 - 200C~ The Pollowing chloroa~lsne3 were ur~ed to prsp~r~ tha rav~raed-phasa:

1. DimethylchlDroRilane-C125i (CH3)2 2. Di-n-butyldichloro~ilane-C12Si(C~Hg)2 3. Msthyl-n-decyldichlorosilane-C125i(CH3~C10H21 4. Methyl-n-octad2cyldichlorDsilarle-ClSi (CH3~C1~ H37 5. n-Octsdscyldichlorosilane-C135iC19H37 ThE following expsriments illustrate the haemoperfusions of haernoadsorptions euccessfully carried out with Lichroprep RP-18 ~ :

Cartridqe structure A small plastics tube of polypropylens with a screw cap lid (SsrYte~dt Co.) W8S used as the mini-csrtridga for the invitro experiment. The tube was 6rm high and 1.5 cm in diameter and held 8 ml. An adapter was '~

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fitted in the lid through a drilled hole. The adapter served to connect the SMA flow-rated pump tube (Technicon In~trumental Oorporation, T,arrytown; New York). The supply to the flexible Technicon tubP-~wss through in~usion systsms. So that the perfusion medium cnuld bs collected up, the bottom end of the small pla8tic8 tube wa9 drilled through and provided with a fo~m ~$1ter. The fo~m ~ilter compri~ed 4 - 5 cross-linked polyethylene layers with pore diameter3 of 0.2 mm to 2 mm.

Human plasma, obtained by centrifuging sevsral units of storad blood, wrs used a8 the oarrier solution for the pharmacons.

Three grams Lichroprep RP-18 ~ wer~ placed in a small gless tube with ~n opening st the bottom. In order to achiavæ a homogeneous watting of tha adsorbent with tha blood/plasma, ethsnol/~qua tide~t. wa~
mix~d 1: 2 with the adsorbsnt. The contants wera uigorously shaken manually several time3. Ths adsorbent thus prep2red wa~ filled into the cartridge Tha ~olvent flthanol/aqua bidest.drippsd through the ~ole at the bottom of the tube. Thro~gh the dripping of the soluent the ad~orbent was sedimented homogeneously nnd tightly oompact.
Tha c~rtridge with the ndsorbent wa3 then ~lushsd through with about 200 ml aquQ bidask. in order to wash out ony remaining solvant~

Tha pharm~con~, emitriptylin, quinine, digoxin, dlgitoxin, methequslon,phenobarbitone nnd phenytoin were used~ w0ra aach dissolved in a toxic conoentration in a litre of pl2sma and vigorously shaken manually sevar21 time~.

The flexible Technicon tubes wsre str2tcheo by a precision roller pump ~.

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(AutD ~nF-lys~r prsportioning pump, T~chni50rl In~trument~al Corporl3tion"
'r~rrytown~ N~w York~. The ~upply tubo~ ~rom tha pl~3ma ~dere pn~e~d through 8 water b~th o~ 37 I;D

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In order to determinæ thP stsndard dsviation, fiv~ 0~p~riments wer~ carri2d ~ut with flve cartridges ~or esch pharmacon. Thess c~rtridgss wer~ perfusad with th~ pl~ema-ph~rmecon solution at a ~low rate of 1 ml/minO Tha overall running time was 1aO minutes.
Thus 100 ~1 solut~on flowed through e~ch rartridge.

To determine the concentration~ of the pharm~cons uoed, specimen~
were t~k0n from the five parallol c~rtridge~ bPtore (P0) end ~ter three-hour parfusion (P1 to P5)o Additional perfusinn experiments were carriad out with heparinized fresh blo~d in order to determine th~ biocDmpatibility r~garding the coagulation param2ters and the cellul r constitu0nts of the blond as well Q~ with purc pl~sma (without pharmacons) to determine the blocompatibility r0garding tha non-callular constituents o~ the blood.

The ~ollowing parametars were thereby examinedO

Elactrophors~i~, coagulation par~meter (TP2, PTT, TZ ~nd ~ibrinogen), colloido3motic prsssure9 pH, HC039 erythrocyte count, h~emoglobin, haematocrit, averags corpuscular volume, 3versge corpuscular haemoglobin concentration, leucocyte count9 thrombocyte count as well as sodium, pota~sium, chloride, calcium, inorganic phosphat~9 iron, glucose, urea9 creatinine, uric acid, overall protein, albumen, bilirubin, cholasterol ond triglycerides.

Determination methods 1. Amitriptylin Amitriptylin was determined with an enzyme immunoassay (Syv~ Co., Darm~tadt) with 0n anti~erum sen~itive thereto. The linearity of the measuring process was in the r~nge from 0.2 to 1 ~g/mlO Dilution o~ the specimens , was carried out with native sarum. Thz lower measuring ranga with this procsss Wa9 O- 1 ~g/ml, snd the upper range 1 ~g/ml.

2. Quinine-HCl Protein wa5 removed from the specimen~ by trichloroQcstic aciJ
and ths clsar sxcass sat to pH 12 by eoda lye and thsn measured apectrophotometrically with a wavs length of 250 nm. The procs~s msasured linesrly in the rQngs from 0.6 to 20 yg/m 3. Digoxin Digoxin was determined with e radlo immunoasaay (aecton Dickinson Kit).

4. Digitoxin Digitoxin wa~ slmilarly Pxamined with radio immunoas~ay (Coat-A-Gount-Digitoxin DiagnD~tic Products Corporstion).

5. Methaqualon Ths apscimens wsre aat to pH 10 by buffer eolution and then extractsd with dichloromethana isopropQnol 9:1- Ths solvsnt Wa8 ramovad and the re~idue absorbed in 100 ml methanol. Ga~ chrom~togr0phy wa~
carried out wlth an OV-17 column at 290 C~ The linear mea~uring rangs was ~rom 0.1 to 20 ~g/ml.

fi. Phenobarbitone PhEnobarbitone wes dst~rmined spectralphotometrically according to the Zak methods with a spsctral photometer Pys-Unicam PU 0~00 UV/vis ~rom ''!~

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Philips, An aliquot psrt o~ the chloro~orm phase ~eparated off was rs-~xtractsd with soda lye. The bsrbiturates thereby chsnge into sodR lye ~nd r~n be tr~csd by subsequent spectralphDtometric msaauremsnt.
The adsorption maximum o~ the barbiturates was 253 to 255 nm in 0.5 N
80da lye (Clin. Chzm. 1973, 615).

7 Phenytoin ~etermining the phenytoin concentr~tion wa9 carr~ out by the fluore3c~nc~
pol~rizotion immuno as~sy techniquo (TDx-system, Abbott-Di~gnostics Division, Wiesbad~n)~

e ults:

Pharmacons The Tabla gives the mea~ured values ~or the pharmacons qx~mined.
Tha T~ble cont~in~ the cDncantrations b~ore perfusion (P0) and after the perfusions (P1 to P5~ of e~ch pharmacon. The cl~ranc~
values ~nd percentage elimination are also shown. Th~ clsaranoa W8~ calculated sccording to the following formula:

r Concentration 1 rConcentrotion L b~fore perfusionJ - ~ after p~r~usionJ
Clearonce (ml/min) _ ~ x Flow rat~
Concentration before perfusion ~2~

Teble:

Pharmacon ,Conc,l Po ' P1 P2 P3 P4 P5 P SDl Avsrage Clearance Eliminotion ~ , (ml/min) in % of initi Amitryptilinug/mll 10.6l'~û.1~0.1 <0.1 ~0.1C0~1¦C0.1 to.o5 1 0.999 99.06 quininelug/mll a.6l 1.51.7 1.2 0,01.5j0.96 0.08 1 o.~ea 88.a4 Digoxin ,ng/ml~ 4.01c0.15 ~0.15~0.15 ~0.15 ~O.lY~0.15~0.05~' 0.96 96.33 Digitoxin Ing/mlj 46.9lc2.0 ~2.0 ~2.0 ~2.0 ~2.0 ,~?- ~0.41~ 96 95.74 Methsqualon'ug/mll 17.21 0.2 0.1 0.1 0.1 0.1 '0.12 0.04 1 0.99 93.30 Phanobarbltana mg/dll 96.4l 1.B 2.2 1.8 1.8 1.~2'1.8a 0.23 ¦ 0.979 Y6.04 Phsnytoin lug/ml! 50.5l 0.69 0.02 0.03 0.27 0.33l0.26 0.27 1 0.99J 99.S8 P0: Concentration bafore per~usion P1 5 Concentra~ions after perfusion P: Mean value of tha concentrations after perfusion SD: Standard deviation * : Standard deviation of the examination methodology Clearance: Po ( 1... 5) - x flow (ml/min) wherein the flow amountad to po 1 ml/inin a~AmitriptYlin:
r With en initial concentretion o~ 10.6 ~g/ml the concentretion after pErfUsiOn W85 bæneath the trnce limit of 0~ 0.05 ~g/mlD

The average clearance value calculated amounted to 0.99 ml/min with a flow of 1 ml/min. The percsntage elimination on aversge was 99% of the total amitriptylin.

The initial concentration P0 emounted to ~.6 ug/ml. The mean value P of the concentrations after perfusion was 0.96 ~ 0.03 ~g/ml.

With a flow of 1 ml/min, an average clenrance of 0.39 ml/min wa~
calculatad, as well as en averaga percentage alimination of 88.84%
of he quinina.

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The initial concentratiDn P~ ~mountsd to 4.0 ~g/mlO Digoxin could no longer ba tr~ced after per~uaion. The lower traceable limit was at 00147 ng/ml. Thua tha average cle~rance amounted to 0.96 ml/min.
with a ~low of 1ml/min, and ths aVeraQe percentage elimination wa~ 96.33%.

The initial concentration P0 amounted to 46.9 ng/ml. Digitoxin could no longer be traced after perfusion/ The lower traceable limit W98 at 2 ng/ml. With a flow of 1 ml/min the auerage cle~rance waa determined at 0.96 ml/~in and the avarage percentage alimination at 95.74 e~ Methaqualon:

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With sn initial cDncentrstion P0 of 17.2 ~g/ml the concantrations ufter perfusion wsre on average 0.12 ~ 0.04 ug/ml, which onrresponds to an avsrage clssrance oF 0.99 ml/min, with a flow of 1 ml/min and to en avsrage perssntags elimination of 93.30%~

f~ Phenolb0~biton2:

With an initial concentratlon P0 of 96.4 mg/dl the concentrations ~ter perfusion were on averege at 1.88 ~ 0.23 mg/dlO Thus en aver2ge clearance of 0.98 ml/min W88 cslculated with e f1DW of 1 ml/min, snd an averaye percentage elimination oF 98.04% ot` the entire phenolbarbitone.

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Ths initial concentration P0 amDunted to 50.5 ~g/ml. The mesn vslue P
of the concentrstion~ ~fter perfusion ~mountsd to 0.26 + 0.27 ~g/ml.

Wikh a flow of 1 ml/min9 an aver~ge cle~rance of 0.99 ml/min was calculated 0æ well ag an average percentage elimination of 99.58%
of the phenytoin.

~iocompatibility.

The p~rameters mentioned on page 4 were determined.

a) Celluler constituents of the blood The erythrocyte count, haemoglobin and derived parameters remain prectically unchanged.

The thrombocyts and lsucocyte counts decreased during perfusion.
In the case oF the thrombocytes, the drop was between 91.5 and 97.7~
and in ths case oF the leucocytes between 37 and 44.4% of the originsl values.

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The lab~r~tory chsmical p~rsmet~rs remain eubat~ntially unch~ng~d.
The tluctuatiDns wers within the standard deviatinns of th~ laboratDry~

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Claims (3)

1. Preparation for haemoperfusion or haemoadsorption characterised in that it consists of silica gel.

2. Preparation according to claim 1, characterised in that the silica gel is a reversed-phase siliceous gel.

3. Preparation according to claim 2, characterised in that the reversed-phase siliceous gel has a pora diameter of about 10 nm and a particle size of 40 - 60 micrometers with a substantially non-polar hydrophobic surface.