CA1039274A - Diagnostic agent - Google Patents

Abstract

ABSTRACT
A diagnostic agent for intravascular administration, consisting of or containing a suspension of minute particles and a radioactive substance is provided, said particle having a size in the order of magnitude of 0.1 - 300 µ m. The suspension is more stable than those previously available, and is not as subject to sedimentation. The particles of the suspension decompose in sufficient time to avoid the serious consequences arising from a prolonged blockage of the blood vessels. In addition, the suspension is more resistant to heat and fluctuations in temperature.
The agent consists of a suspension of minute particles which comprise a polysaccharide built up of glucose units or a physiolog-ically acceptable derivative of said polysaccharide in a physiolog-ically acceptable aqueous liquid. The particles comprise a water-insoluble, but hydrophilic, swellable, three-dimensional network of molecules of the polysaccharide, or of the derivatives thereof, cross-linked by means of bridges having bonds of a covalent nature.
At least one radioactive substance is included in the agent and is bound to and/or enclosed in and/or exists in mixture with the minute particles. The network may be broken up by x-anylase in blood plasma into water-insoluble fragments within a time after injection that can be varied within wide limits.
The suspension is prepared by suspending the minute particles in a physiologically acceptable aqueous liquid.

Description

:. . Th~ present in~ention relates to a d:Lagnostic ~gene rO r ~ ~
in~r~vascular admini-qtration, said agent cv~isi~t~ng of or oon- I .
;: taining a suspension o~ minute particle~ and a radioact~q ;;~ .
: ~ sub3tan~e. ; . ~ ~ : ~
- 5~ : It ii3 previously known to use suspensions of minute,. ` ~ ;
.;pre~erably spherioal partic~es of different materials labelled ": lwith radioactiv~ isotopes for intravascular administration to .anlmals ~nd humans ~or diagnostic purposes. Particles pra~rably~ :
. used for this purpose are prepared ~rom protein suGh a9 serum albumin. Such radioactiye serum albumin particles are described or example in the German Published Spec~fication No. 1,916~704~ .
~; ~, ; Particles baised on polysaccharides or on synthetic polymers (e.g. polystyrene~ and ev~n on inorganic material~ labelled ~ith ,radioisotopes have~also been used experimen~allr for intra~
. ;15~ soular administration in te.sts carried out on animals. ;
The particles pr~vious~rtested ~n th~s regard are. en~
: ............ ,........ , . , . 1: . ..
bered wi~h a number of disadvantag0s, One isuch diisad~antage. ; i ~ .
,. .. , , , , .; j ~ .
- : . rei~iide~ in the. fact that some partic~es do not decompoise or ~dacomposG ~oo slowly ih the blood ~essels and remain more or :~ :
~-. 20 i lesg permane~ltly in sa~d v3i~3sel9. The.y oan g~re r~se to small ; thrombose~ which do not regreisiBj e~en should the particles.be subsequently dis~olved or decomposed snd lea~o the ~lcod ~a~sel `~
38tlo~ whi~h obviou~ly leads t.o seriou^ ~onsequen~e~6 :' ~ ~ . ; ,'` . .'- ' ' ,, ' ' - , , ' ', .

392~7~a ` ` Anothfar di~advantag~ resides in the fact that mo3t of th~
- previou~ly ta~ted- particles, for example albumin-based partiolas, ;;..~ exhibi.t poor suspension stability and are pro~e to sadimentation .' ~ ' '' '.and/or conglomeration (e.g. owing to the high speGific weight .;
'' 5 ; ; and/or the adhesivsness of the particles) rendering it neee~sary~
.. . '- ~ to sub~ect the suspension to ultrasonic treatment in order to. I
~ ' prevene this ~rom happening.' Howfsver, the stability of fsuch' i~. . '. ..;earlier partlcle suspensions treated ultrasonically ~ 8 ~er~
poor:and the ~uspension must be used a~ soon as possible after ~' I0'~ aid treatment. 'The stability of the particle~ (for example ' the albumin particles) i9 orten SO poor as to render i~ necassary~.
to store 3aid~particlas in '~reeze dried condition, the durab11it~
.' o~ bhe particles being,ne~ertheless, still ~imited, Some. " `~
.Y pàrt~cles are'unable to withstand ~ariations in tempe'rature .': :
~;15.'.'j~:and aannot be sterilized by heat treatment. The prevlously ~ "~
. '',~ ';;'tested particles hà~e eikher not been dissolvable or degradabla ~
. in blood plasma, or have been dissol~able or"degradable only l ~ .
' '' in an irregular and non-reprodufcable manner, or have been ohanged'~
'.f'.in'thi~ regard during ~torage, which present~ ¢ons~derable 1; "
~'2'0 ;'''~:dlsad~antage8'and ri~ks. .' " '.
'I0 has bsen surprisingly discovered that the aformentioned' di8advantages encountered with the previoUf~yus~d partlclee can~
'. j'.'9be el1minated by means o~ ~he present invention. . : ' . ' ..
~ ' The present in~ention relates to a diagnostic agen~ whlch '.'~.... '~ ~25~ intended to be adminstered in~ravascularly and whlch consist9 oft or ~ontains a su~pension of minu~e particles~ preferably ha~ing~ a ~ parti¢le size ln the order O t magnitude O t 0.1 ~ 300 ;~
um (m~crometer) j ~omprising a polysa¢Gbar~de b~ilttt~ Up, 0~' "^'.~.~ .. ' ,'.'; "~':~ '.' Ct 2 ~t ~ t ~

:: glucose units, or iQi phy~iologlcally acceptable derivatiYe o~ .
aid polysaccharide in a physiologically acceptable aqueou~ :
.
liquid, said ~uspen~ion also comprising a r~dioacti~re 8ubstanc~ .
;` . which is bound to and/or el~closed in and/or in m~x'c~re ~r~th .: ~5 . .. ~aid mtnut particles. . -. ` The diagnostic agent according to ~he inven~ion ~3 charac-. .` ;~: terized in that.the aforementioned particles comprise a water~
.- :. . lnsoluble but hydrophilic, swellable (i.e. swellable in water~
three-dimensional network of molecules of the polysaocharide o~ ~r ~ tha -deri~atlve thereo~ cross-Iinked by bridges ha~ing bonds.o.f a ~ovàlent ~ature,.said network being degradable by x-amylase n blood plasma into water-soluble ~ragments~ either directly ~.
or subsequent to a preceding splitting-off of possibly occurring .
. ,~. ' substituents, pre~erably glucoside bound and/or ester bound `.
I5 ,;~.substitue,nts~ in the polysaccharide by the action o~ an en~yme, ;
; `.I, . .~ ~ preferably glucosidase and/or esterase ~ in blood plasma.0 ` . ;
. . The poIysaccharide whichlis built up.of giucose unlts .::
~ ;;and which shall be incorpora~ed(as such or in the ~orm of a ; . .'. physio10gica1ly acceptable derivati~e) in cross-linked rorm ln ...
20.~ the par~1¢les, shall be capab1is o~ being degraded by a-amy1a8e :: ~.i; in~o:wat~r-soluble ~r~gments, i.e. the poly~ac¢harid~ shall ' con~ain ~ 4) glucosidic lin~ages which ar~ hydr~l~zable :~
. . by a-amylases. ~xamples o~ such polysaccharides include p¢im~ly~tarch and glycogen or dextrins thereo~. The star~h . 25..~ m~y be amylose or amylopectin ~r mixturo~ ~hereof. Other . g1uoose conta1ning polyqaccharides which aan be hydrolysed by a-amglase Gas~ also be used~ in ¢on~ec~io~ with whi~ aid poly~

.. . .. . ~ '~ , ! ` , , . '' i :' ' '' ~r~, . - :

~ 39 Z~ 4 accharides m~y ba synthetic or may be obta$ned from biological , material, for exampIe from microorganisms. It ~s simplest and :
cheapest, however, to use starch in th~ form o~ amylo~e or amy~pectin or mixtures thereof. SimilarlyJ ~he phy~iologieally ~ ~ 5; , acceptable derivative of the polysaccharide shall be degradable - -~by -amylase directly or subsequen~ to a precsdi~g spl~tting-o ` o~ substituents under the action of an enzyme ~n blood plasma, such as ~or example estera6as or gluco~idases. Substibuant8 ~;in the polysaccharide may, for exa~ple, be hydroxyalkyl group~`
io~ (which are option.ally broken by one or more oxygen at~m~ or.
~example lower hydroxyalkyl groups ha~ing for example 2 - 6 carbon atoms such a~ 2-hydroxyethyl, 2-hydroxypropyl and/or .2j3-dihydroxypropyl; and/or alkyl groups, e.g..lower alkyl ~. ;
b . `:. groups having 1 - 6 carbon atom~ such as methyl and/or ethyl, .~.
:;15 ;i .:and/or subs~itoted alkyl groups, e.g. sub~tituted with carboxyl i ~
;groupa such as carboxy methyl and/or alkanoyl groups, or substi ~.
; ~ tuted alkanoyl groups, e.g. lower alkanoyl groups haYing e.g.
, . ` . 2 -:6 carbon atoms, such as acetyl, propionyl, 2-hydroxyp~opanoyl~
; :~; ;succinoyl and/or glutaroyl. The reducing end gr~up of the poly-~ s~c¢haride may be unchanged or modified. For example, it may .
; ;`~,~ be ox1tiqed or redûoed, 90' that sald end of the polysaccharlde ~:
: ohain ie terminated wi~h a carboxyl group or a primary hydroxyl ;~
i group~- It may, for example, al o be pres~nt in tha form or a, ,``!`
gluooside, e.g. with an alcohol such a~ glycerol.
~, 2,5~' The cros~-llnking bri~ges may be bound to the ~olecule~
; ` o~ the p~lysaccharide or the derivative ~hereof ovcr different ~typa~.of bonds. In accordance with a particularly sultabl~
: ; , èmbodime~t qi the i~entio~ e~e bo~ds are.é~her bo~d8 . ' ;....... ' , ' ''''' , " ''; .'"' '. ' , '; ,', ~ 2~ !
In accordanoe with a f urther suitable embodimsnt o~ tha inven- .
ion/ ~aid bonds are'ester bonds, the term ester bond~ bein~ , used here in its widest significance, Thu~, the term al90 ~. ~ .include~ for example, carbamic acid es~er bonds and th~ocarbam~ . :5 i, ;acid ester bonds. Preferably, aliphatic b:rldge bu~ld~ng lin~s ~' are chosen, although said links may al~o be~ for ex~mpla, aroma~a ;,;.. ,,~ ;.','~or a~aliphatic.
~, ..` ~,;~., .' The cross-linking bridges ma~ also con~ain to ~d~ant~g9~ ,.', hydrophilic groups, e.g. hydroxyl groups ~e.g. one to 5iX; ' ~' i,~10 ,~,,,hydroxyl,groups.in each bridge~. ............................... , ' `
.'~.; . ' ' In accordance with the invention, the cross-linked poly~
?~ ~, saccharide molecules in the practically infinite'., thiee-d~men~
i`'.. ~ional networ~ m,ay be substituted with other substituents ~han ; the cross~linking bridges. For example, these substituents ; 'i.
,15 ,,,~may be,one or more o~ the aforementioned substit~ent~, e.g.' :,,,:,',,~,'hydroxyalkyi, alkyl and/or alkanoyl. As will be,readily ~nder~
,, ,:'. ,.. stood, monorunctionally bound substituents or~ginating from ., .. ' ',,,,.' the cro~s-linking agen~ may also occur.
~ `' ' In~accordance wi~h a particularly suitable and practical ;' 20, .' embodiment of,the ~nventi~n, the molecules of t,he polysacoharlBe .
.' ~"': or o~ the deriva~ive thereo~. are cross-linked by mean3 of .bridgas'whioh ar~ bound ~o these,moleoules o~er ether bonds, .: , ;' wherein the.bridges between the ether bond~ may adra~tageou~ly, :,:~
be ~traight or branched aliphatic saturated hydrocarbon chains ; ;~-:25 :~ whioh are substituted by one or more hydroxyl group~ (~'.g. one .to 9iX~ hydroxyl groups) and wh~ch contain 3 - 30..carbon atom~
pre~rably 3 ~ 20 càrbon a~om~, su~h a~ 3 - 10 cerbon,~tom8; `
and wht~h~are optio~ally bro~en by one or more~oxygen ~tO~d a 5 i .

, ~ Z7 (e.g. one to six oxygen a~oms). Examples o~ such ether-bound cross-linking bridges are -CH2 . CH(OH) . CH2- and -CH2 . CH(OH) . CH(OH) . CH2-and -CH2 . CH~OH) . CH2 . O . CH2 . CH(OH) - CH2- `,.
and 1--CH2 . CH(OH) . CH2 . O ~ (CH2)n . O . CH2 . CH(OH) . CH2- , I ~
.where ~ is an integer, for example an integer from 2 to 4, , ~ ~ :
and CH3 -CH2 . CH(OH) . CH2 . O . ~H . CU2 . CH2 . O . CH2 . CH(OH).CH2-and -CH2 . CH(OH) . CH2 .O.CH2.CH2ØCH2.CH2ØCH2.CH(OH).a~2-and -CH2 . CH(OH) . CH2ØCH2.CH(OH).CH2Ø(CH2)nØCH2.CH(OH). :~ :
.CH2ØcH2.cH(OH)-cH2~
where n is an integer, for example an integer from 2 to 4.
In accordance with another embodiment of the ln~ention, ~
the molecules of the polysaccharide or of the derivative there- -:
. .
of are cross-linked by means of bridges which are bound to 2a sa~d molecules over ester bonds which may preferably be carboxy- ~ ;
lic acid ester bonds,but which may also be carbamic ~cid ester bonds or thiocarbamic acid estsr bonds, the bridges betwesn the~ :
.; -ester bonds advantageously being s~raight or branched aliphatic saturated hydrocarbon chains containing 2 - 20 carbon atoms, `.
preferably 2 - 10 carbon atoms such as 2 ~ 6 carbon atoms, and being optionally broken by one or more oxygen atoms (e.g. one to six oxygen atoms) and optionally substituted with one or more hydroxyl groups (e.g, one to s~.x hydroxyl group~

. ~
- 6 ~

,.

~39~7~
Examples of such e~ter-bound tin ik~ wideæt signifieanc~) cross-linked bridgas are -0 . C0 . (CH2)n . C0 . 0-, where nl is an integer/ for example an integer from 1 to 20, preferably 2 - 10 such as 2 - 6, and -0 . C0 . CH2 . 0 . CH2 . C0 . 0- and -0 . C0 . NH . (CH2~n . NH . C0 . 0- and -0 . CS . NH . (CH2)n . NH CS 0-, where n2 is an integer, for example an integer from 2 to 6.
In accordance with the invention, the three-dimensional network in question is capable of being degraded by -amylase in blood plasma into water soluble fragments, either directly .:: : . .
or subsequent to a preceding splitting-off of possibly existing substituents in the polysaccharide under the action of an enzyme in blood plasma, for example, esterases or glucosidases.
The degradation of the network by a-amylase takes place owing to the fact that a-amylase hydrolyses glucosidic linkages in the polysaccharide chains of the network. In order that the network should exhibit suitable properties with regard to the degradation of said network by ~-amylase, it is generally suitable that the substitution degree of the polysaccharide with respect to the cross-linking bridge substituents and possible occurring singly bound substituents, which cannot be split-o~f by en~ymes in blood plasma, is lower than 70 per cent) pre~er-ably lower than 60 per cent, said substitution degree being given as the percentage of the number of substituted glucose units with respect to the total number of glucose units prese~t.
For example, said substitution degree may be lower ~han 55 per cent, e.g. lower than 50 per cent~ It is generally suitable for the substitution degree of the polysaccharide with respect to the cros~-linking bridge subs~ituents and possibly~oc~urring .

.

singly bound substituents, which are not capable o~ being split-off by enzymes in blood plasma, to be higher than 1 per cent, preferably higher than 2 per cent, sa~d ~;ubstitutivn degrse being given as the percentage of the number of substituted glucose units with regard to the total numb~r of glucose units pressnt. For example, the substituti~n degree may be higher than 5 per cent, ~or example higher than 10 per cent. ~ -Generally, the substitution degr~e with respect to all kinds of substituents (i.e. the total substitution degree) is suitably ;
lower than ~0 per cent preferably lower than 70 per cent, for exampla lower than 60 per cent and suitably higher than 1 per aent, preferably higher than 2 per cent, for example higher than 5 per cent. Thus, for example, the substitution degree may be 35 per cent, i.e. o~ 100 gluco~e unitf3 in the polysaccharide chains 35 of these glucose u~its are carr~ing at least one substituent. I
In accordance with the inYention, the cross-linked poly- ~ -sa~charide product is insoluble in water but swellable in water to a gel. It may, for example, contain more than 50 per cent by weight o~ water, such as more than 60 per cent by wsight of water, preferably mor~ than 65 per cent by weight of water, for example more than 70 per cent by weight of water~ It may, for example contain less than 99.g per cent by weight of water, preferably less than 99.5 per cent by weight of water, such 25 as less than 99 per cent by weigh~ of water, generally less than 9~ per cen~ by weight of water, such as less than 95 per-cent by weight o~ water. -,. fi~

I

~ ~ ' ' ' ' ' ~ ~3 ~
In accordance with the invention, the mesh size of the three-dimensional network may be such that protein molacules of the same order o~ magnitude as a-amylase are able to pene- ;
trate into the particles in their water-swollen condition.
The mesh size can be determined with the aid of conventional gel chromatographic tests, using substances, such a~ protains, o~ dif~erent molecular sizes.
In accordance wqth the invention, the three-dimensional network of the particles may be such that said network is broken up more slowly b~ ~-amylase in the outer layer o~ khe particle than in the inner part thereof. In this case, the three-di~ensional network of the particle may exhib:Lt a hi~her sub~titution degree of cross~linking substituents and/or mono-functionally bound substi~uents in the outar layer of the par-ticle than in the inner part thereof.
The particles may hare an irregular shape or may be spherical. Preferably, spherical particles are chosen.
Preferably, the particles have substantially a particle size of the order of 0.1 - 300 ~m (micrometer), e.g. 1 - 100 ~
in water-swollen state. When it is desired to clog fine blood vessels, particles having a size of 5 - 60 ~m ln water-swollen state are often chosen.
In accordance with ~he invention9 the particle ~ize can be ~elected so that said particles clog fine blood vessels located in or leading to a selected portion of tha bod~, ~ub~e-quent to being administered intravascularly.
- ~ In this instance, the particle si~e is selected in ` ~ depen~qnce upon the dimensions of ~he blood vessels ~o be clogged., :

.

3l~a39Z~
:,.
An example of ~ine blood vessels of interest in this context is blood capillaries having a diameter of about 5 - 15 ~m and metaarterioles having a diameter of about 15 - 300lum. In certain instances, for example when deterrnining the volume o~ ~
blood using radioactive particles, the se]Lected size of the ;;
particles may be such that the particles clo not fastenj even in the finest of blood capillaries.
One advantageous embodiment of the invention is chara¢-terized in that the three-dimensional network can be degraded by a-amylase into water-soluble fragments havlng substantially a molecular w~ghtbeneath 50,000. In this way, ~he major .
portion of the fragments are excreted over the kidneys with the urine.
In accordance with the invention, the meshes of the three-dimensional network may be enlarged subsequent to cross-linking by partially degrading said network, a.g. by partial hydrolysis o~ glucosidic linkages in the cross-linked polysaccharide ~ -chains. Such a partial hydrolysis may be effected, for axample, with an acid or a-amylase.
In accordance with the invention, subsequent to being in-jected in~o the blood vcssels, the particles can be degraded into water-soluble fragments by a-amylase within, ~or example, the space of some few seconds to many hours, depending upon ~ the effect desired in each individual caset With ~regard to the particles according to the invention, the degradation time may thus be varied within wide limits and can ba well ~nd reproduc-ably established for the desired field o~ use.

.' .. , .

. . . ` ~

~ 0~ ~ ~ 7 The cross-linking of the polysaccharide molecules to a practically infinite three-dimensional network can be eYfected by reac~ing the polysaccharide or the polysaccharide derivative in question wnth an at least bifunctional cross-linking a~ent.
Preferably, the cros~nking agen~ is reacted with hydroxyl groups in the polysaccharide chains whereby many bridge~ of t~e following type are obtained between the polysaccharide chains:
Pl - O - B - O - P2 wherein - B - is a bridge-forming link between oxygen atoms derived ~rom hydroxyl groups in two diffe-rent polysaccharide chains Pl and P2. Preferably, the bridge-forming link B contains at least 3 carbon atoms, for example 3 - 30 carbon atoms or 3 - 20 carbon atoms.
For the purpose of obtaining cross-linklng bridges which are bound to the polysaccharide chains over ether bonds, the polysaccharide or the p~ysaccharide derivative can be reacted for example, in an alkaline aqueous solution with a c~o~c-linki~g agent, for example of the type:
y X . Al . Z (I) and X A2 Z (II) where X, ~ and Z each represent a halogen atom, pre~erably chloro or bromo and Al and A2 each represent a straight or branched aliphatic, saturated hydrocarbon chain which is substituted by one or moro hydroxyl groups (e.g. one to six~ and which prefer ably con~ains 3 - 30 carbon atoms, for ~xample 3 - 20 carbon atoms9 such as 3 - 10 carbon atoms and which is optionally broken by one or more oxygen atoms (o.g. one to six), or with a corresponding epoxide compound which can be obtained ~rom the the c ~ ound (I) or (II) by splitting off hydroge~ halide, ~l~139~
Examples of bifunc~ional substances of the formula X . Al . Z
and corresponding epoxide compounds which can be obtained from compounds of said formula by splitting off hydrogen halide are;
CH2 - CH - CH2 . O . (CH2)n . ~ . CH2 . C~l - CH2 where n is an integer, for example from 2 to 4 and CH2 - CH . CH~.O.CH2.CH2ØCH2.CH2.0-CH2 CH ~ 2 and ,CH3 CH2 - CH . CH2 . O . CH . CH2 . CH2 . O . CH2 . CH - CH2 and C ~ - CH . CH2 . O . CH2 . C~ ~CH

and CH~ - CH CH2 . O CH2 CH~OH) . CH2 . . CH2 . CH - CH

or corresponding halogen hydrins, and bifunctional glycerol deriv~tives of the formula X . CH2 . CH(OH) . CH2 . Z, for example, dichlorohydrin and dibromohydrin, or corresponding epoxide compound (obtainable by splitting off hydrogen halide) of tho formula CH2 - CH . CH2 . Z, e.g. epichlorohydrin or epi-~ 0~
bromohydrin. Another example of such a bifunctional compound is - ~
1,2 - 3,h-diePoxYbutane of the formula CH~ - ca . CH - CN2. ~ -An example of a trifunctional cross-linking agent (which is an epoxide compound corresponding to a compound of the -~ ~ormuI~' Y )is ' X A2 ' Z

. . !
, ' lX -,1 :

: :'.~ ` .' .

1~13~7~
H2 . ~ CH2 . CH . CH2 . O . CH2 . CH - CH2 CH2 . CH -CH2 ~ 0~ :
The polysaccharide or the polysaccharide derivative is reacted with such a quantity of an at least bifunctional cross-linkin~ agent that a water-insoluble gel is formed, iOe. a practically infinite three-dimensional network which exhibits the desired properties. One skilled in thi~ ar~ can rleadily establish empirically a suitable relationship between the quantities of different polysaccharides or polysaccharide deri-vatives and cross-linking agent.
For the purpose of obtaining cross-linking bridges which are bound to the polysaccharide chains over ester bonds, the polysaccharide or the polysaccharide derivative can be reacted in a manner known per se with, for example, aliphatic or heterocyclic or aromatic dicarboxylic acids or reactiva deriva-tives thereof, e.g. with dicarboxylic acid dichlorides (e.g. of succinic acid or of adipic acid) or ~or example, with diisocya-nates or diisothiocyanates. Other cross-linking agent~ may also be used, The cross-linking reaction, in addition to bridge-building,~
also o~ten results in the introduction of monofunctionally bound (i.e~ singly bound) substituents (mono-ethers, mono-esters etc.) . -from ths cross-linking agent, iOe. only one reactive group in the at least bifunctional bridge-building agen~ has raacted with a hydroxyl group in a polysaccharida chain whilst the other reactive group or groups in the bridge-formi~g agent ha~e e.g.

:

.

.

instead reacted with, e.g. water to form, eOg~ hydroxyl groups or carboxyl groups, etc. Consequently, the polymer product most frequently presents also monofunctionally bound substitusnt~
originating from the bridge-building agent; e.g.
-O . CH2 . CH(OH) . CH20H when the bridge-building agent i9 epichlorohydrin, and -O . CH2 . CH(OH) . CH2 . O . ~CH2)4 ~ O .
. CH2 . CH(OH) . CH20H when the bridge-building agent is 1,4-butandiol diglycideether or, e.g. -O . CO . (CH2)n . COOH
when the bridge~building agent is a dicarboxylic acid dichloride.
The polymeric gel product can be obtained in particle ~orm elther by producing the polymer in kho form of large pleces (bulk polymerisatlon) and thæn disintegratin~ said product, e.g. by grindingj or by producing the product by bead polymeri-sation techniquss in the form of spherical particles. In this latter case, the reaction mixture is dispersed to droplet form `;~
in an inert liquid which is immiscible ~herewith, wherea~ter the gel particles formed by th~ reaction in the droplets are recovered. Particles having a spherical shape are preferably used. The desired particle size can be obtained by fractionating~
the particles, e.g. by screening the same.
The gel product obtained can be substituted wlth different ~;
groups, e g. for controlling the rate with which the particles ;
are degraded by -amylase in blood plasma~ For this purpose, hydroxyl groups in the polysacch~ride chains may be substituted with ~ubstituents, e.g. of the aforementioned type such as lower alkyl, lower carboxyalkyl, lower hydroxyalkyl and~or lower alkanoyl. Thæ substituents may, ~or example, be ether bound and~or ester bound to the polys;~charide chains.

.

For the purpose of controlling the rate of degradation of the gel particles in blood plasma, the particles can be subjected to partial hydrolysis in vitro (e.g. with an acid or with a-amylase) prior to or during the preparation of tha suspension.
This partial hydrolysis of glucosidic linkages is continued until the gel particles have obtained the desired properties.
In accordance with the invention, at least one radioàctive substance is included in the diagnostic agent. This agent has the form of an intravascular administratable substance. For exampla, it may be a radioactive isotope of an inert gas such as xenon or ~rypton, or a substance which contains a radio-active isotope of iodine or phosphoru~, such as sodium iodide or sodium phosphate, or a substance which contains radioactive technetium, e.g. sodium pertechnetate, or e.g. a substance which contains a radioactive isotope of chromium, indium, gold, yttrium, ytterbium, cerium, cobalt, carbon or hydrogen.
A large numb~r of such substances containing radioactive isotopes suitable for the intended purpose are known to those skilled in this art. Two or more dif~erent radioactive isotopes may also be used. The radioactive substance or substances are present in the diagnostic agenk in a concentration and a degree o~
radioactivity which is sufficient to enable the relevant diagno-sis to be made.
In accordance with the invention, the radioactive substance or substance3 may ba in mixture with the minute particles. Tha substance may, e.g., exist in the ~orm of extremely minute, insoluble particles (eOgO of the same size or smaller ~han the pol~accharido-based particles)~ optionally on an inorganic or ..

; ~ - 15 -~ 9 ~7 ~
organic carrier material The radioactive ~ubstance may ~lso be a water-soluble substance. It may be dissolved in the physiologically acceptable aqueous liquid in the su~pension.
In this respect, conventional radioactive qubstan~os for in~ra-5 vascular use are normaly used In general, however, the radioactive substance is bound to and/or enclosed in the minute particles. For the purpose of enclosing the radioactive substance in the particle, a radioactive substance, which is insoluble in the aqueous sus-pension liquid or has a very low solubility therein, may beprecipitated out in the swollen gel particle or suspended in the ~ction mixture when the polysaccharide ls cross-linked to a three-dimensional network. In this case, the radioactive substance may also be bound to an insoluble inorganic or organic carrier, which may be adsorbed on the particlss.
Conver.tional methods may be used for binding the radio-active substance to the particles, for example whilst utilizi~g ~ ;
the hydroxyl groups of the particles.
The radioactivity o~ the particles may originate from radioactive technetium, which can be readily incorporated in the particles with a high degree o~ labelling and with very slight or no leaking o~ soluble radioactive material, by adding to an aqueous suspension of the particles radioactive sodium pertechnetate and a reduction agent such as stannous chloride, ~5 sodium thiosulphate or sodium dithionite in one or more steps.
The diagnostic agent is administered in quantities suffi-cient to enable, in each individual case, the desired effect to be obtained. In general, the quantity o~ agent administered ..
~;y - .

~ 16 -~ 2~
(calculated for each individual) corresponds to 0.1 to 2,000 mg of particles, e.g. 0.5 to 200 mg of particles and is dependent upon the examination to be carried out, e.g. ~he region of blood vessels to be examined and possibly to be cl~ggedO For example, the quantity may be in the range of from 0.001 mg to 50 mg, preferably 0.01 mg to 25 mg, for example 0.05 mg to 10 mg o~ particles per kilogram of body weight.
The concentration of the particles in the suspension may be varied within wide limits, depending upon the intended use.
For example, it may be more than 0.01 mg, e.g. more than 0.1 mg~
such as more than 1 mg of particles per 1 ml of suspension, and e.g. less than 200 mg, e.g. less than 50 mg, such as less than 25 mg of partioles per 1 ml of suspension. Ths physiolo-gically acceptable aqueous liquid in wh~ch the parkicles are suspended may be liquids normally used for intravascular in~ec-tion, (e.g. physiological sodium chloride solution, i.e. 0.9 per cent aqueous solution of NaCl) or aqueous solution3 of the salts occurring in the blood plasma. In some cases gluoose or sorbitol solutions, e.g. 5 per cent aqueous solutions thereo~, may be used. Other phy~iologically acceptable substances may be added to the suspension, e.g~ sucrose or dextran.
Preferably, sterile suspensions of the particles are used.
Sterilization can be effected by heat treatment e.g. autoclaving, or by adding substances which prevent the growth of microorga-nisms. The suspensions may also be prepared aseptically.
The diagnostic agent is intended to be administered intra-vascularly, i.e. pra~erably in blood vessels, although it may also be admini~ster~d, fo~ e~ample, in the lymph ~es~els.
' -:

~39 Z~
In accordanca with one embodiment of ths in~en~ion, the particles of the agent may clog the finer vessels subsequent to intravascular administration of said agent, thereby to cause the M ow of blood in the vessel to be impeded, so that the retention time o~ the radioactive substance in the vessel system in question is prolonged or the passage travellsd by said substance redirected. When ths diagnostic agent is administ~ed, the particles of the agent and the radioactive substance are preferably held in the same portion of the blood vessel and preferably upstream of the finest vessels, as seen in the ~low direction.
In accordance with another embodiment of the invention, the radioactively labelled particles of ~he agent are smaller than the diameter of the finest vessels, thereby enabling the lS diagnostic agent to be used, ~or example, to measure the rate of ~low of the blood and the blood volume. These measurements may be performed in a ma~ner known E~_ se.
By means of the present invention it is possible to satis-factorily fill a vessel system or a portion of a vessel with a radioactive substance with a prolonged retention time of the radioactive substance in said vessel portion or the system in question, in a manner which is ~ree from risk, owing to the faYourable properties of the particles, inter alia the soft gel consistency of the particles, and owing to the fact that the three~
-dimensional network o~ said particles is water-swollen and that the rate at which the particles are degraded enzymatically into water-soluble fragments, can be varied in a reproducable and determlnable manner, which can be controlled precisely both in vitro and in vivo, (This is in co~ra~t ~o previously known ,.

~ ~39 ~
particles, including albumin microspheres, which are digested irregularly mainly by phagocytosis in vivo. Currently used albumin particles are not significantly digested in cell-free body fluids.) ThusJ with the diagnostic agent according to the invention it is possible, in comparison with previously known methods, to obtain improved and new diagnostic results in a manner ~ree from risk.
The invention also relates to an auxiliary age~t for use when preparing the relevant diagnostic agent for intravascular administration comprising minute particles, prsferably having a particle size in the order of magnitude of 0.1 - 300 ~ , con-sisting of a polysaccharide built up of glucose units, or a physiologicall~ acceptable derivative of said polysaccharide.
The auxiliary agent according to the invention is characterized in that the particles consist of a water-insoluble but hydro-philic, swellable, three-dimensional ne~work of molecules of the polysaccharide or derivative thereof cross-linked with bridges . .-having bonds of a cov~ ~t nature, said network being d~gradabls by -amylase in blood plasma into water-soluble fragments direct-ly or subsequently to a preceding split~ing-off of possibly occurring substituents, preferably glucoside-bound and/or ester-bound substituents, in the polysaccharide by the action of an enzyme, preferably glucosidase and/or esterase, in blood plasma.
The disclosures made in the aforegoing with respect to the minute particles in conjunction with the diagnostic agent al6l0 apply with respect to the particles of the auxiliary agent.
The invention also rel;~tes~ to a method of effecting a diagnosis with the aid of radioactive substances, in which a diagnostic agent is intravascularly administered which con~ists : , ~

1 ~3 ~ 2~
of or contains a suspension of minute particles, preferably particles having a si~e in the order of magnitude o~ 0.1 to 300 ~m, consisting of a polysaccharide built up of gluco~e units or a physiologically acceptable derivative of said poly-saccharide in a physiologically acceptable aqueous liquid, saidsuspension also comprising a radioactive subs-tancs which is bound to and/or enclosed in and/or exists in mixture with the ~ ;
mi~ute particles, whereafter the radioactive ra~diation is ~ ;
measured ovsr the relevant body portion or on a sample takan `~
from said body (e.g. an intravascularly taken sample).
The method according to the inventiGn is characterized in that the particles comprise a water-insoluble bu~ hydrophilic, ~wellable, three-dimensional network of molecules of the poly-sacoharide or of the derivative thereof cross-linked b~ bridges which have bonds of a covalent nature, said network being degradable by a-amylase in blood plasma to water-soluble frag-ments directly or subsequent to a preceding splitting-off of possibly occurring substituents, preferably glucoside-bound and/or ester-bound substituents, in the polysaccharide, under the~
action of en enzyme, preferably glucosidase and/or esterase, in blood plasma.
The disclosures made in the aforegoing with respect ko the minute particles etc. in conjunction with the diagnostic agent and the auxiliary agent also apply with respect to the particles etc, in conjunction with ~he me~hod of effecting a diagnosis.
Particularly favourable results are obtained with the method ac-cording to the invention owing to the favourable prop~rties o~
the particles. ~ ~
The invention will now ba illustrated by means of a 3 number of examples.

~ 20 -~ 2~7 Examp,le 1 333 g of soluble starch having a molecular weight ( ~) of approximately 20,000 were dissolved in 533 ml of water containing 53 g of sodium hydroxide and 2 g of sodil~ bor~hydride. Suhse-quent to being stirred for four heurs, the solution was allowedto stand for two days wi~h a layer of octanol on the surface thereof (about 0.5 ml). A clear solution was obtained.
In a cylindrical reaction vessel provided with a thermo~
meter, a cooler and agitator there were dissolved 20 g of Gafac(R) PE 510 (a complex organic phosphoric acid ester which served as an emulsion stabilizer and which is obtainable from General Aniline Film Corp.) in one liter of ethylene dichloride at room temperature, whereafter the previously prapared starch solution was added. The mixture was stirred at a speed su¢h that the water phase was dispersed to droplet ~orm of the desired magnitude in the ethylene dichloride phase. The size of the droplets formed upon agitation of the starch suspension in ethylene dichloride was controlled with the aid of a micro-scope. After adjusting the speed of the agitator to 1100 rpm, which gave an average droplet size of 70 ~ , 40 g of epichloro-hydrin were added.
After a reaction time of 16 hours at 50C, the product was poured into 5 liters of acetone and allowed to settle.
The supernatent liquid was drawn of~ and the product was slurried in ~ 5 liters of acetone9 The acetone was drawn off) ~ liters o~ water were added and the pH adjusted to 5, by adding acetic a¢id. The product was then slurried a further 4 times in ~ liters of water and five timesrin 5 liters of acetone, where-after the produ¢t was dried in vacuum at 50C for two days~
` The produc~ weighed 241 g.

~ 21 ;3Ç92~
The polymer particles were insoluble in water but swelled in water to gel form, the gel particle~ containing ~3 per cent by weight of water The degree of substitu~ion was abou~ 35 Part of the product was suspended well in water~ The suspension was then screened by water-streaming on screens having a mesh size of 100/um, ~0/um, 56 ~m~ 40 ~m and 25 ~ .
The particles remained on the different screens in accordance with the following weight distribution ~he weight are given in dry weight);
Mesh size in ~ weight (g) .
~0 7.9 ~0 4.9 :~
11.2 15 The fractions were washed with di~tilled water, and were then :~
washed free of water with aoetona and dried in a vacuum at 50C for ~wo days.
Example 2 With respect ~o products prepared in the manner disclosed in Example 1 but with varying quantikies of epichlorohydrin, ~he sffect of the quantities of epichlorohydrin used, on the degra-dation of the particles by means of a-amylase was examined in the ;~
following manner:
7 mg of particles having a size which, when wet-screening the particles in accordance with Example 1, passed through a screen having a mesh size of 40/um but which remained on a screen having a mesh size of 25 ~ , were weighed in a polypro-pylene vessel and slurried in 20 ml of 0.05 M sodium pho~phate .

~ ~2 ~

; . . . : ....... 1~ " .

~ ~3 ~
buffer, pH 7, wi-th 0.05 % Tween(R) 20 ~wetting agent) (polyoxy-ethylene-sorbitan-monolaurate from Atlas Chemie GmbH~. The ~eaker was placed under agitation in a bath, ~he temperat~re of which was ad~usted to 37~C. When the t~3mperature had stabi-lized, there were added 200 ~1 of ~-amylase from swine pancreas ~rom a stock solution having a concentration of 150,000 IE/l or 24,000 IE/l (IE - international units). 500lul of sample were pipstted at uniform intervals down in Ellerman tu~e~ containing

2 ml of an 1 per cent aqueous sodium hydroxide solution, where-after the tubes were centrifuged for 5 minutes. One ml of thesupernatant was then pipetted over to a plastic tub~, for deter-mining the quantity of substance which , as a result Or the ~f~ect o~ the ~-amylase) had been released ~rom the particles and had passed into solution.
As a measurement of the rate of degradation, the time was recorded in which hal~ of the mass of the particles was refound in the supernatant. The following res~ was obtained:
Epic~lorohydrin Water Degree of Time (min) Time (min~
(quantity in g~ content substitution with 240 IE with 1500 I~
of swelled (in ~0) a-amylase/ ~-amylase~
~article ~% weight) ~ 96 ~ 5 ~3 2S 93 ~ 20 19 ~ :
~5 29 26 ~, 5 ~3 36 3~ 15~ 5 ~0 40 50 21 76 42 73 ~ 30 .

, ~ 23 -.. - ; .

:
~39~
When the amount of epichlorohydrin was 60 g J only 25 %
of the mass of the particles passed into solution in two hours with 240 IE -amylase~l.
~m~ : ' 1.0 g o~ dry particles produced in accordance with Example 1 but at an agitator speed of 1500 rpm and having a ~ wet-size which, when~screened, passed through a screen having a mesh size of 40/um but remained on a screen having a mesh size of 25 ~ , were swollen in 30 ml of water. 0.4 g of acetic acid anhydride dissolved in 5 ml of tetrahydrofuran was added drop-wise to the particle suspension over a period of 10 minutes (the pH being kept at ~.5 - 9 by addition Or 1 M aqueous NaOH
solution), whereafter the suspension was neutralized. The gel grains were then washed with distilled water and acetone, and 1~ ~hen dried. The water-swollen particles contained approximately ~5 % by weight of water. The total degree of substitution was about 50 %.
Hydrolysis with 0.1 N sodium hydroxide and titration with 0.1 N hydrochloric acid gave 1.51 mmol of acetyl per gram of dry product. When degrading with a-amylase in acoordance with the method descrlbed in Example 2, hal~ of the mass of the par-ticles were found in ~he supernatant after 6 hours with 240 IE
~-amylase per liter and after 1 hour and 9 minutes with 1500 IE
~-amylase per liter, respectiYely. For the unsubstituted star-ting product, hal~ o~ the mass of the particles was found inthe supernatant after 40 minutes wlth 240 IE ~-amylase per liter and a~ter 15 min with 1500 IE a-amylase per liter, respectively.
Thus, tha substltution with acetyl groups had considerably in~
: ', ~ ~ 2~ ~ ;
;, ., - .

-~)35~Z~;~4 ~ `

creased the degradation time in tha presence of a-amylasa in vitro.
E~mple 4 ~4 g of carboxymethyl starch having a substitution degree of 20 % and a molecular weight ( ~ ) of about 20,000 were dissolved in 3~ ml of water containing 13.7 g of sodium hydroxide and 0.05 g of sodium borohydride~ Subsequen~ to being agitated for four hours, ~he solution was allowed to stand for 2 days with a layer of octanol on the surface thereof (somefew drop~
A clear solution was obtained.
In a cylindrical reaction vessel provided with a thermo meter, a cooler, and an agitator, there were dissolved 20 g of Ga~ac(R) PE 510 (a complex organic phosphoric acid ester which serves as an emulsion stabilizer) in 265 ml of ekhylene di-chloride at room temperature, whereafter the previously preparedstarch solution was added. The mixture was agitated at a speed such that the water phase dispersed to droplets of the desired s~ze in the eth~lene dichloride phase. The size of the droplets formed in the starch suspension in ethylene dichloride upon saàd agitation was controlled with the aid of a microscope. Subse-quent to adju~ting the agitatin~ speed to 1500 rpm, 10.3 g of epichlorohydrin were added.
After 1~ hours reaction time at 50C the product was poured i~ 2 liters of acetone and allowed ~o settla. The super-natant was drawn off and the product slurried in 2 liters ofacetone. The acetone was drawn off, 2 liters of water were -addsd and the pH adjusted to 5 with acetic acid. Th ~roduct was slurried 4 times with distilled,water admixed with 0~5 g of , : :

a,03~;~ r sodium azide) and 5 times with 1250 ml of acetone, whereafter ~he product was dried in vacuum at 60C for 2 days. The product weighed 69 gO The particles were insoluble in water but swelled in water to gel particles, the particles containing about 90 % by weight of water. When degraded with -amylase in acoor-dance with the method described in Example 2, half of the ma~s of the particles was found in the superna~ant after 4.5 and 2.5 hours respectiv~ with -amylase content 240 and 1500 I~/l respectively.
~xam~le 5 2 g of dry particles were prepared in the manner described in Example 1, but with an agitating speed of 330 rpm and swollen particle size which passed through a screen having a mesh size of 125 ~m buk which remained on a screen having a mesh size f 100 ~ . The particles were stirred in 25 ml of 0.1 M hydro-chloric acid at 20C. A sample amounting to about 0.3 g of particles was taken at different intervals of time, said ~amples ;
being centri~uged and washed with distilled water 3 times and treated with acetone and dried in a vacuum at 50C for 16 hours.
The time taken for h~Lfthe mass to degrade to water-soluble fragments under the action of ~-amylase as described in Examplo 2 w~s then determined. The following results were obtained:
Time for h~drochloric acid Degradation time (min) treatment ~hours~ , w_th 1500 I~ ~-am~lase/l o 60

3 52 ~-" ~ ,' `'';"~ ' ' ''~ ., ' ~ _ ~6 ~

: . .
- :

~ 3 Example 6 16 g of a dry product prepared in accordance with Example 1 ha~ing a particle size which, when wet-screened, pass~d through a screen having a mesh size of 40 ~ but which remained on a screen having a mesh size of 25 ~ , were swollen and ~uspend~d in 400 ml of dis-tilled water. 0.~5 g of propylene oxide was added and the pH adjusted to 12 with 2 M sodium hydroxlde. The sus-pension was maintained at 50C and agitated for 24 hours, whereafter the suspension was neutralized with acetic acid9 washed with water and wet-screened with water. The fract;ion which passed through the screen having a mesh size of 40 ~ bu~
which remained on a screen having a mesh size o~ 25 ~ was recovered. 2.5 g product was obtained. The product was insoluble in water buk swelled in water to gel particles, said particles containing approximately ~0 % by weight of water.
Thet3tal degree of substitution was h~ %.

Example Z
An experiment was carried out in the manner disclosed in Example 1, but instead of epichlorohydrin, there were added 90 g of 1,4-butandioldiglycidyl ether and the speed of the agi-tator was maintained at 1400 rpm, which resulted in an average droplet size of 25 ~ . In other respects the experimental con-ditions were the same as those disclosed with reference to Ex~mple 1 and washing and drying were also ef~ected in the manner disclosed in Example 1. 2~4 g of product were obtained.
The product was insoluble in water, but swelled in water to gelp articles, the particles cpntaining about 75 % by weight watërO~(~The degree o~ substitution was estimated to be about 40~.) ;,, , , ~ 3 ~ 7~
10 g of the product were s~ pended in about 200 ml of water and were subjected to an ultrasonic treatment process. The suspension was then screened by water-scre~ning through screens having mesh sizes of 56/um, 40/um and 25lum~ The particles remained on the different screens in accordance wi~h the follow-ing weight distribution (the weights are given as dry weight):
weight (g) l~0 2.

4.2 The fractions were washed with distilled water and acetone, whereafter they were dried.
xample ~
33 g of hydroxyethyl starch having a molecular weight ,`
( ~ ) of about 143,000, were dissolved in 54 ml of water con-taining 5,3 g o~ sodium hydroxide and 0.2 g of sodium borohydride.Subsequent to a clear solution being formed there were added 2 g of GafactR) PE 510 dissolved in 100 ml of ethylene dichloride and the mixture was agitated at a speed such that a suspension of droplets having an average ~iameter of 50 ~ was formed.
4 g of' epichlorohydrin were then added and the mixture was skirred for 16 hou~s at 50C, The product was poured into ace-tone and allowed to settle. Thé acetone was decanted and the produot swollen in water. The pH was adjusted to 5 with HCl, wherea~ter the product was washed wi~h distilled wa~er, acetone ¦~
and petroleum ether.
The product was then dried at ~0C in vacuum. The product weighed 33.6 g and presented a substitution degree of about 66 ~o.
The water-insoluble product swelled in water to gel particle , r , ~

a ,.

~ ~3 ~ 2 form, the particles containing about 75 % by weight of water~
10 g of the product were screened on screens having a mesh size of ~0/um, 56/um, ~0/um and 25/um by water screening. The particles remained on the different screens in acoordanc~ wi~h the following weight distribution ~dry weight):
Mesh size (~m) weight (g) ~ ; ~
gO 3.9 56 1.5 0.9 1.5 Example ~
4.5 mg of dry, spherical water-insoluble po~ymer particles (having a size when swollen of 25 - 40 ~ ) prepared in accordance with Example 1 were swollen and suspended in 3 ml of an isotonic sodium chloride solution. In this wa~ ~he suspension contained 0.9 million of swollen particles having a diameter of abou~ ' 25 -40 ~ . 0.5 ml of acidified stannous chloride (5 mg of stannous chloridetml 0.1 N HCl) and 2 ml of sodium pertechnetate (1.36 mCi~ were added to the suspension. After 10 minutes reaction time at room temperature, the suspension wa centri-fuged, whereafter the radioactivity of the particle mass and of the supernatant sas measured individually in a radiation detector.`
It was found that the particle mass contained 1.24 mCi, whil~
the superna~ant contained 0.12 mCi, i.e. the labelling degree wa~ 91 %~ Subsequent to washing the particles in a 0.9 %
~odium chloride solution, the labelling efficiency was studhed chromatographically. This involved applying about 10 ~1 of the prepare;d suspension on a thin layer chromatographic plat~ (Merck ~ 29 -103g2r74 silica gel) for the separation in rnethyl ethyl ketone. No traces of free, unreduced Tc-99m could be discovered. The sus-pension was also examined in a B~rker calcula~or chamber undsr a microscope. No conglomeration of the particles could be observed - this in contradistinction to previously known par-ticles which have a pronounced eonglomeration tendency. ~ -Example 10 100 mg of spherical particles (having a particle size when water swollen of 25 - 40 ~ ) prepared in accordance with Example 1 were labelled with sodium pertechnetate in the manner de~cribed with reference to Example 9, but with varying quanti-ties of stannous chloride and hydrochloric acid. The following labelling degrees were obtained:

Stannous chloride 1 ml 0.05 ml 0.01 ml containing two 0.1 M 0.1 M 0.~1 M
~ hydrochloric hydrochloric ~ydrochloric crystal water (mgJ acid acid acid 99 97 ~9 17.5 95 92 92 ~,5 9~ 95 9~

5.0 ~7 90 ~7 - `

~xample 11 100 mg o~ sphQrical particles (having a particle size when water swollen o~ 25 - 40 ~ ) prepared in accordance with Example 1 were suspended in 5 ml of stilled water. 50 mg sodium di-thionite and 10 ~1 of an aqueous sodium pertechnetate solu~ion eonta~ning 0.5 mCi per 10 ml were added to the suspension.
After one half hour reaction time at room temperature (20C), the product wa3 washed and centrifuged and the labelling degree ~ 30 -.

was measured in the manner described in Exampla 9. A labelling degree of 90 % was obtained.
Example 12 An anesthetized rabbit was placed in a recumbent po~ition on its back beneath a gamma camera. One m:L suspension of par-ticles (500/uCi) prepared in accordance with ~xample 9 and corresponding t~ abvu~ 300,000 Tc-99m-labelled polymer particles were then injected into the vein of the right ear. Registrations were made at regular intervals. Immediately after the injection, the lungs were visibilized in a pronouncedly satisfactory manner with the possibility of anatomic detailed examination. After appr~ximately 10 minutes the liver, kidneys and bladder could also be visibilized to a certain extent, owing to the fact that the a-amylase enzyme of the body had begun to degrade the polymer. After 30 minutes, the test was terminated by registering ~ -tha activity of separate organs, t~ere being refound in ~he lungs ~9.6 %, in the liver 6~6 %, in the kidneys 3.4 % and in the muscle tissue 0.4 %. During the whole of the registration time, the lower lobe of the right lung could be sati~factorily visibilized, due to low absorption of radioactivity by the li~er.
Example 13 A dog weighing 30 kg was catheterized via the groin artery, the end of the catheter being placed in tha livsr artery and the position of said catheter being verified by X-ray examination. 300 mg of particles produced in accordance with Example 1, which particles9 when wet-screened passed through a screen having a mesh size of 56 ~ but which remained on a zcrecn haviLg L mesh s~ze of 40 ~ ~ were suspended in 20 ml ~ 3~ ~ 2~ ~
of physiological sodium chloride solution, whereafter 1 ml of a sodium per~echnetate aqueous solution containing an activity of 0.2 millicurie Tc-99m was added. This mixture was introduced -;
into the vessel bed of the liver via the catheter. The radia-tion over the area of the liver was measured with a radiationdetector using so-called rectilinear scanning, and the radiation intensity over different portions of the body portion was photo-graph~oally regiskered via an oscilloscope. The photographic image showed the anatomic ex~ension of the vessel bed in greater ~;
detail. After approximately 20 minutes further measurements were made. The image obtained showed that ~he gamma-radiating isotope Tc-99m had now disappeared from the organ in question~
and the test could now be repeated with similar results, which i5 of great value in respect of the physiological studies of ~`
experimentally produced changes in the regional blood distribu~
tion. The method can also be used for the differen~ial diag-nosis of tumours.
xample 14 0.3 g dry, spherical, in water insoluble but swellable particle9 ~ith a swelled diameter ~ize of 20 - 40~) prepared according to Example 1, but with 50 g of epichlorohydrin instead of 40 g mentioned in said example (the ~aber-swollen particles contained approximately 75 % (by weight) o~ wa~er and the total degree of substitution was hO %), were swelled and suspend~d in 10 ml of a solution of stannous chloride ~10 mg SnC~ , H20) in 10 ml of 0.01 N deaired hydrochloric acid. This mixture was then poured into 10 ml of 1 M sodium acetate-acetic acid buffer of pH 5Ø After a reaction time of 3~ min., the mixture ~ - 32 1~3~2r~9!~ . , was cen~rifuged and the centrifugate was washed twice with deairad distilled water. After this the particles were suspen-ded in 100 ml of a deaired 10 ~0 saccharose solution and 0.1 ml deaired 1 M sodium acetate-acetic acid buffer of pH S~0 was added. Aliquotes of 1 ml of this suspen~sion were portioned into 5 ml bottles, which were sealed under nitrogen. After addition of 1 ml sodium pertechnetate (1 5 mCi Tc-99m~ml) and shaking for 15 min, sample bottles were centrifuged, and the supernatant was wi~hdrawn, whereafter the radioactivity of the particle mass, the supern~n~ and the bottle were measured individually in a radiation detector. It was found that the particle mass contained 1.45 mCi, while the supernatant contained 0.03 mCi and the bottle 0.02 mCi, i.e. the labelling degree was 97 %-A dog weighing 20 kg was anaesthetized with pentobarbital (30 mg per kg body weight) by intravenous injection through a brachial vein catheter. A gamma camera was adjusted to detect Tc-99m activity over the lungs. The Tc-99m labelled content of a bottle (1 ml, 3 `mg particles/ml, 1.5 mCi~ was injected via the brachial vein catheter. Tc-99m activity in the lungs was immediately observed on an oscilloscope. Sequential photographs of the image of the lungs on the oscilloscope were ~ ;
taken with a Polaroid camera attached to the oscilloscope.
A detailed and clear perfusion lung image was obtained immediate- -ly following the injection. The~image persisted long enoughfor a thorough examination of both lungs.

~m~a~
3 mg dry, spherical, in water insoluble bu~ swellable , , ~ ~ .

3L~)3~
particles (with a swelled diameter size of 20 - 4G ~ ) prepared according to Example 1, but with 50 g apichlorohydrin instead of 40 g mentioned in said example (the particles containing approximately 75 % (by weight) of water and the total degree of substitution was 40 ~0), were swelled and suspended in 1 ml of 0.1 M acetate buffer of pH 5.0 and autoclaved in a sealed bottle.
0.1 ml of acidified stannous chloride (1 mg SnC12, H20~1 ml O.01 N hydrochloric acid) was added to 1 ml sodium pertechnetate (2.3 mCi Tc-99m) 9 using aseptlc technique (millipore fi:Lters).
Af~er thorough shaking this solution was added aseptically to the suspension of particles. Af~er 10 minu~es reaction time '!~
at room temperature the suspension was centrifuged and the labelling degree was measured as described in Example 9 using aseptic technique. It was found to be 9~ %. The particles were then suspended in 1 ml 5 % sterile glucose solution using aseptic technique, and injected as a bolus into the right cubi~al vein of a man aged 65. He was suffering from pulmonary embolism affecting the lower lobe of the right lung. The lung perfusion was vi~ualized by a gamma camera, positionsd to cover the chest `~
and upper abdomen. Excellent scans wero recorded from several positions, allshowing impaired perfusion in the area embolized.
Repeated ~cans could be p~rformed during 3 hours, with no detect-able radioactivity in the liver, thus permit~ing clos~ examina-tion of the diseased lung section~
Previously used albumin based particlss usually give high li~er acti~ity soon after injection, ~ interfering with visualization of the anatomically close righk lung while the par~icles ~n this example gave no disturbing liver activity when~ed as a diagnostic agent ~or the lungs.
.~ ~
. ~ ' . .
~ 3ll ~

.

.

Claims (11)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of preparing a diagnostic agent for intra-vascular administration which method comprises binding a radio-active substance to or enclosing said substance in or both binding it to and enclosing it in minute particles which par-ticles are substantially spherical in shape and in water-swollen state have a size within the range of 0.1-300 µm and which par-ticles comprise a water-insoluable but hydrophilic, swellable three-dimensional network of molecules of a polysaccharide selected from the group consisting of starch, glycogen, dextrins thereof and physiologically acceptable derivatives thereof cross-linked by means of bridges which are bound to these molecules over ether bonds and which bridges comprise straight or branched aliphatic saturated hydrocarbon chains substituted with one or more hydroxyl groups, said chains containing 3 - 20 carbon atoms and optionally being broken with one or more oxygen atoms, the total substitution degree for the polysaccharide being lower than 70% and higher than 5%, said substitution degree being given as the percentage of the number of substituted glucose units with respect to the total number of glucose units present, said three-dimensional network being degradable by the .alpha.-amylase in blood plasma into water-soluble fragments either directly or subsequent to a preceding splitting-off of possibly occurring substituents in the polysaccharide by the action of an enzyme in blood plasma and said cross-linked polysaccharide product swelling to a gel in the presence of water, said gel containing more than 50% and less than 99.5% by weight of water.

2. A method according to claim 1, wherein the polysaccha-ride molecules are also substituted with substituents others than the cross-linking bridges.

3. A method according to Claim 2, wherein the other substituents are at least one member selected from the group consisting of 2-hydroxyethyl, 2-hydroxypropyl, 2,3-dihydroxy-propyl, acetyl, propionyl, 2-hydroxypropanoyl, succinoyl and glutaroyl.

4. A method according to Claim 1, wherein the total sub-stitution degree of the polysaccharide is lower than 60% and higher than 10%, said substitution degree being given as the percentage of the number of substituted glucose units with res-pect to the total number of glucose units present.

5. A method according to Claim 1, wherein the cross-linked polysaccharide product swells to a gel in the presence of water, said gel containing more than 65% by weight of water and less than 98% by weight of water.

6. A method according to Claim 1, wherein the mesh size of the three-dimensional network is such that protein molecules of the same size as .alpha.-amylase are able to penetrate into the particles in their water-swollen state.

7. A method according to Claim 1, wherein the three-di-mensional network of the particles is such that said network is broken up more slowly by .alpha.-amylase in the outer layer of said particle than in its inner part.

8. A method according to Claim 1, wherein the three-di-mensional network of the particle presents a higher substitution degree of cross-linking substituents and monofunctionally bound substituents in the surface layer of the particle than in the inner part thereof.

9. A method according to Claim 1, wherein the three-di-mensional network can be broken by .alpha.-amylase into water-soluble fragments having a molecular weight beneath 50,000.

10. A method according to Claim 1, wherein the radioactive substance contains a radioactive isotope of technetium.

11. A diagnostic agent for intravascular administration, consisting of or containing a suspension of minute particles and a radioactive substance which is bound to, enclosed in or both bound to and enclosed in said particles, which particles are substantially spherical in shape and in water-swollen state have a size within the range of 0.1 - 300 µm and which particles comprise a water-insoluble but hydrophilic, swellable three-di-mensional network of molecules of a polysaccharide selected from the group consisting of starch, glycogen, dextrins thereof and physiologically acceptable derivatives thereof cross-linked by means of bridges which are bound to these molecules over ether bonds and which bridges comprise straight or branched aliphatic saturated hydrocarbon chains substituted with one or more hydroxyl groups, said chains containing 3 - 30 carbon atoms and optionally being broken with one or more oxygen atoms, the total substitution degree for the polysaccharide being lower than 70% and higher than 5%, said substitution degree being given as the percentage of the number of substituted glucose units with respect to the total number of glucose units present, said three-dimensional network being degradable by the .alpha.-amylase in blood plasma into water-soluble fragments either directly or subsequent to a preceding splitting-off of possibly occurring substituents in the polysaccharide by the action of an enzyme in blood plasma and said cross-linked polysaccharide product swelling to a gel in the presence of water, said gel containing more than 50% and less than 99.5% weight of water, when said diagnostic agent is made by the method of claim 1 or by its obvious equivalents.