AU680828B2 - Islets of Langerhan's in pure form - Google Patents

Description

4' .4 OPI DATE 15/03/94 AOJP DATE, 09/0,6/94 APPLN. ID 47099/93 PCT NUMBER PICT/EP93/02178 AU?34T099 (51) Internationale Patentklassifikation 5 (11) Internationale Verdiffentlichungsnummer: WO 94104169 A61K 35/39 Al (43) Internationales Veroffentlichungsdatum: 3. Mff rz 1994 (03.03.94) (21) Intertiationales Aktenzcichen: PCT/EP93/02 178 Verdiffentlicht A'it internationalein Reclwrehenbericljt (22)1 lnternationales Anmeldedatum: 16. August 1993 (16.08.93) M~it geiderten Anspriichen und Erklidrung.

Prioritfitsdaten: P 42 27 066.9 16. August 1992 (16.08.92) DE (71)(72)Anmelderund Erfinder: HERING, Bernhard, J. [DE/l DEJ; Friedrichstr. 35, D-35392 GieIgen (DE).

(74) Anwijlte: ZUMSTETN, F. usw. Br~uhausstr. 4, D-80331 Mtinchen (DE).

(81) IBestimmungsstaaten: AU, CA, JP, US, europfiisches Patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

(54) Title :LLANGERHANS' I1N PURE FORM (54) Bezeichnung: LANGERHANS'SCHE TNSELN IN REINER FORM (57) Abstract The invention relates to preparations of transplantable Langerhan3' islets in particularly pure form, a novel production method and the therapeutic and/or diagnostic use of the islets.

Zuwammenfassung Die Erfindung betrifft Zubereitungen von transplantierfilhigen Langerhans'schen Inseln in besonders reiner 'Form, ein neues Herstellungsvcrfahren sowie die therapeutische und/oder diagnostische Anwendung der Inseln.

2 ISLETS OF LANGERHANS IN PURE FORM TECHNICAL FIELD This invention concerns the preparation of isolated Islets of Langerhans with a purity level higher than 98% relative to the volume of purified cellular particles and a manufacturing process for such a preparation, as well as the diagnostic and/or therapeutic application of the preparation on a human or animal body.

BACKGROUND OF THE INVENTION Since the introduction of insulin to diabetes therapy by Banting and Best in the year 1922, the ketoacidotic coma of diabetics with lethal outcome has become quite rare. The treatment with insulin has, however, not prevented the surfacing of diabetogenic secondary complications in the form of microangiopathy, such as to the eyes, kidneys and nervous system, as found in the vast majority of diabetic cases.

As of 1966, efforts have been made to alleviate generally known limitations of insulin therapy through 20 the transplantation of a healthy pancreas. However, the risks of a surgical procedure, particularly the required life-long immuno-suppressive treatment for the prevention of a reaction of bodily rejection on the part of the recipient, do not, in the opinion of prominent 25 diabecologists, justify a pancreas transplant as a procedure of choice.

There has long been an effort, rather than transplanting the entire organ from a brain-dead organ-donor's pancreas (such as an accident victim), rather to isolate the insulin-producing Islets of Langerhans and to transplant those only, whereas their introduction into the Vena portae (liver portal vein) is the most prominent type of application. It is known from numerous tests on animals that intra-portally transplanted islets invade the end branchings of the portal vein, absorb insulin production depending upon requirements such as blood sugar level, correct the diabetic metabolism and prevent the life-threatening Si23228A s ~-cll°e P-~9 3 complications of diabetes. It is further known from numerous tests conducted on animals that, with the use of low level immunogenic islet trans-plantation, the latter, as opposed to pancreas transplantation, requires no, or only temporary, immuno-suppressive treatment of the diabetic recipient.

There is evidence of islet transplantation in diabetic humans as well. Only one female patient has so far been free of insulin-use for a period of almost two years, after islet transplantation (Warnock et al., Diabetologia 35: 89-95, 1992). Lack of success in many further attempts are explained by the transplantation of islet suspensions of insufficient purity level, i.e.

insufficiently executed separation of non-islet tissues such as exocrine fragments, ductal elements, lymph nodes and vessels. Such islet suspensions with an insufficient purity level are characterised by very high immunogenicity, and, after the transplant, are .o endangered by the frequent appearance of the body's rejection-reactions.

Despite the use of immuno-suppressive therapy protocols, proven to be beneficial in heart, liver and kidney transplants, impure islet suspensions are rejected at an early stage. The focal point of specialists' interest has been cleaning procedures of transplantable islet suspensions, or their preparations, respectively, in a particularly pure form, obtained from donor organs.

In the published PCT-Application W088/09667 a procedure is described with the aim to obtain purified cell suspensions from donor organs such as the pancreas by subjecting cell suspensions, such as suspensions containing Islets of Langerhans isolated from combined tissues, to so-called density-gradient centrifuging. In numerous publications, particularly overviewing articles such as London N.J.M. et al: Islet purification. In: Pancreatic Islet Cell Transplantation (Ricordi C., Editor), R.G. Landes, Austin, Texas, USA, pp 113-123, 1992, the particular methodology of density-gradient S:23228A

I

4 centrifuging primarily based upon variable sedimentation of cells and/or cell aggregates of varying density (also referred to as isopyknic-gradient centrifuging) is described and its application as a cleansing procedure for suspensions with cell aggregates of Islets of Langerhans, whereby purity levels of up to 90% relative to the volume of the purified cellular particles have been obtained. Contamination of hence available islet suspensions with undesirable cellular particles such as exocrine fragments, ductal elements, lymph nodes and vessels may be readily identified under the light-microscope at 100% enlargement.

SUMMARY OF THE INVENTION According to one aspect of the invention, there is provided a preparation of isolated Islets of Langerhans having a purity level of more than 98%, said purity level being relative to the combined volume of the Islets of Langerhans and other cell material in the preparation.

In another aspect of the invention, there is S 20 provided a procedure for the manufacture of a preparation of isolated Islets of Langerhans of a purity level higher than 98% of cell material relative to the volume of the purified cellular particles, comprising: e a) distending the pancreas with a physiologically 25 acceptable liquid and extracting Islets of Langerhans with additional cellular material out of the pancreatic tissue, b) separating extracted Islets of Langerhans from .i additional cellular material through at least one measure of separation primarily based upon varied sedimentation of cell material of varying diameter, and if so desired, c) subjecting the obtained Islets of Langerhans to a treatment which will reduce their potential immunogenicity/antigenicity, and/or preserving the treated islets.

In yet another aspect of the invention there is provided a procedure for the separation of a plurality of Islets of Langerhans from additional cellular material, SS:23228A I the additional cellular material comprising exocrine, ductal, vascular and lymphoid elements, comprising the steps of: distending a pancreas with a physiologically acceptable liquid; extracting the plurality of Islets of Langerhans and the additional cellular material through digestion of the pancreas; separating the extracted plurality of Islets of Langerhans from said additional cellular material by sedimentation based on variation in densities between the Islets of Langerhans and the additional cellular material; and separating the extracted Islets of Langerhans from said additional cellular material by sedimentation based on variation in diameters between the Islets if Langerhans and the additional cellular material.

OV. In a further aspect there is provided a preparation of Islets of Langerhans prepared by a method of the invention.

Preferably the procedure is characterised in that Islets of Langerhans and additional cellular particles are obtained from the comnbired tissue of the distended pancreas by way of enzymatic or possibly mechanical 25 influence.

The pancreas can be distended with the use of a physiologically acceptable fluid, possibly containing collagenase and/or other proteases.

Preferably, the measure of separation, primarily 30 based upon variable sedimentation of cell aggregates of varying diameter, is isokinetic gradient centrifugation.

PrefErably, the isokinetic gradient centrifugation is carried out in a physiologically acceptable aqueous medium with linear density gradient.

Preferably, the isokinetic gradient centrifugation is carried out in a aqueous medium containing polysucrose of molecular weight of approximately 400,000.

The step of separating islets from additional cell S:23228A I 6 material may further comprise the steps of a) providing in a suitable container a bottom layer consisting of colloid and a physiologically acceptable aqueous medium, followed by another layer of medium having in its incorporated volume a continual gradient with a density range of approximately 1.005 to 1.600 grams/cubic centimetre and a viscosity range of approximately 1.1 to 15 cP at approximately 4 0 C to 37°C, and b) adding the suspension of Islets of Langerhans and additional cell material.

Preferably, the measure of separation primarily based upon varied sedimentation of cell material of varying density is the isopyknic gradient centrifugation.

The procedure may be further characterised in that the isopyknic gradient centrifugation is carried out in a aqueous medium with discontinuous density gradients.

Preferably the isopyknic gradient centrifugation is carried out in a aqueous medium containing polysucrose 20 with a molecular weight of approximately 400,000 and 0 sodium-diatrizoate.

The treating step of step described above may comprise: S reducing the potential immunogenicity/antigenicity 25 of the obtained Islets of Langerhans through a follow-up treatment selected from the group of commonly used procedures consisting of culturing, UV-radiation, gammaradiation, incubation with antibodies or encapsulation.

The preserving step may comprise preserving the 30 obtained Islets of Langerhans in the form of culture and/or by cryo-preservation.

A preparation of isolated Islets of Lancerhans embodied by the present invention is characterised by an increased purity level of Islets of Langerhans in comparison to islet suspensions that have only undergone one measure of separation based primarily upon variable sedimentation of cell material of varying density.

The general definitions and terms given above and in S:23228A *a'I 7 the text below have the following meaning in the framework of the invention.

The term "cell material" encompasses cellular fragments or particles, whole cells and agglomerations of whole cells up to agglomerations of cells, also referred to as cell clusters.

The term "physiologically acceptable carrier fluid" defines all aqueous media as described in the respective literature as suitable for cleaning, diagnostic and transplantation procedures for Islets of Langerhans.

Preferred are media commonly referred to as Hank's Balanced Saline Solution (Hank's Solution), Minimum Essential Medium, Medium 199, RPMI 1640, CMRL 1066, University of Wisconsin Solution, etc.

The preparation of isolated Islets of Langerhans can be prepared from human or animal pancreas donor organs, and may be used for diagnostic and/or therapeutic procedures, preferably on humans, but possibly on animals as well.

Accordingly, in another aspect of the invention there is provided a method of treating a mammal for 0e diabetes mellitus, comprising the step of administering a preparation of the invention to the mammal.

i:o.0 For therapeutic procedures for the treatment of 25 diabetes mellitus, both type I and II, the preparation of o° isolated Islets of Langerhans from the human pancreas is preferable. Due to limited availability of human pancreas from donors and the high prevalence of diabetes *mellitus among the population, animal pancreas is used as 30 well; in such a case pig pancreas is preferable. For diagnostic use the preparation of isolated Islets of Langerhans from animals, particularly pig, is preferred.

The preparation of isolated Islets of Langerhans from human pancreas for diagnostic application is another subject of the invention.

The preparation may be in the form of a suspension injectable into the vena portae (portal vein of the liver) or the vena lienalis, the spleen pulpa, beneath S:23228A II IPCIPIIPII P.~L~LI~ uol-~ -8 the kidney capsule (casing), into an omental sack, or into an epiploic flap, by intra-peritoneal, intra-muscular, subcutaneous, intracerebral, or intratesticular injection.

Preferably, the preparation is in the form of a parenterally injectable suspension.

The preparation of purified Islet of Langerhans themselves may have one or more of the following characteristics: an increased purity level of Islets of Langerhans compared to islet suspensions after application of only one measure of separation primarily based upon varying sedimentation of cell material of varying density; a loss of less than 3% islet volume; the diameter of the majority of purified cell material is greater than 100 im; a reduction of immunogenicity by an average of .0 S" a reduction of potential micro-biological contamination; 20 consistency of micro-fluorometric viability S' indication; increase of glucose-stimulated insulin secretion indication in vitro by an average of more than 50%, as well as 25 increase of recovery of the islet volume after one or several immuno-modulated treatments, each compared with pre-cleansed islet suspensions after application of one measure of separation primarily based upon varying 3 sedimentation of cell material of varying density, 30 however, prior to use of a subsequent measure of separation primarily based upon varying sedimentation of cell material of varying diameter; and an amylase content lower than 0.05% in comparison to the islet suspension after release from the tissue of the distended pancreas, but prior to the application of any measures of separation to be performed subsequently.

It is an advantage that the present invention may embody a preparation of isolated lets of Langerhans S:23228A I -M 9 having an especially high purity level. It is a further advantage that the preparation may have reduced immunogenicity in vitro which can be evidenced with the use of a standard test.

BEST METHOD OF PERFORMING INVENTION Procedure Step (a) This step in the procedure is usually known and generally applied during common isolation and purification procedures for Islets of Langerhans.

The term "distension" refers to expansion. For the purpose of distension the pancreatic outlet passage, i.e.

of human or pig pancreas, via which digestive enzymes formed in the exocrine part of the pancreas are released into the small intestine, are cannulated and 2 ml of Hank's Solution are injected through the cannulation retrogradely into the pancreatic passages with approx.

0.1 1.0% collagenase per gram of pancreatic tissue.

This step of the procedure should preferably be carried out according to the method described by Lacy P.E. et al. in Diabetes 16: 35-39 (1967) and Gray D.W.R.

et al. in Diabetes 33: 1055-1061 (1984).

The term "digestion" refers to the digestive processes. In the course of digestion the pancreas is 25 dissociated into Islets of Langerhans and cell material, under enzymatic and mechanical influences. This method should preferably be carried out according to Ricordi C.

et al: Diabetes 37: 413-420, 1988, as well as the method described in the PCT Application WO 88/09667.

The term "protease" refers to enzymes known by names such as neutral protease, clostripain, trypsin, elastase, dispase, etc., besides the known pure types of collagenases.

After completion of Step a) of the procedure we obtain a suspension of the separated Islets of Langerhans in media, as defined above, which are yet impure due to the additional cell material contained.

Procedure Ste (b) S:23228A Y~I~ I 10 The term "measure of separation, primarily based upon variable sedimentation of cell material of varying diameter", refers to a generally known separation procedure, the application of which for the isolation and purification of Islets of Langerhans is new. This measure of separation is treated in international publications under the term "velocity sedimentation", which involves the following procedural variables: sedimentation at 1 gram, counter current centrifuging (elutriation), centrifugation in re-oriented zonal rotors and isokinetic gradient centrifugation. Preferable here is the known procedural variable commonly referred to as "isokinetic-gradient centrifugation".

In one particularly preferred application form of this procedure the isokinetic gradient centrifugation is combined with another measure of separation primarily based upon variable sedimentation of cell aggregates of •varying density.

The term "measure of separation primarily based upon 20 sedimentation of cell aggregates of varying degrees of oat. "density" defines another separation method, usually well known, which has also been applied for the isolation and purification of Islets of Langerhans (see publication by London loc. cit., Page This measure of 25 separation is described in international publications under the term isopyknic gradient centrifugation- A S* detailed description of the preferred method of application is given in the Example.

The pariicularly preferred form of application of S" 30 the procedure is characterised in that: b) the extracted Islets of Langerhans are separated from the additional cell material through subsequently executed measures of separation primarily based upon variable sedimentation of cell aggregates of varying degrees of density and varying diameter.

In another application form of the procedure one may proceed in reverse order, in combining the measures of separation primarily based upon variable sedimentation of S;23228A 1 ~b Pd~ 11 cell aggregates of varying diameter and varying degrees of density.

The isokinetic gradient centrifugation is preferably carried out according to: Pretlow T. G. in Anal Biochem 41: 248-255, 1971, and Pretlow T.G.II and Pretlow T.P. in Cell Separation: Methods and Selected Applications, Vol. 5, Academic Press, pp 281-309, 1987 description of methodology in a physiologically acceptable aqueous medium with linear density gradients. In the publications such a medium is given the term "gradient" and is formed in a so-called "gradient mixer", or in the process of centrifugation itself. The gradient has the highest level of density at the bottom of the centrifuge tube and decreases in density at greater height.

Appropriate media for the formation of gradients are, for example, known under the names of Ficoll®, Percoll® (Pharmacia, Freiburg), and may contain auxiliary substances such as polysucrose, polyvinylpyrrolidone, albumin or dextran. For measures of separation primarily 20 based upon variable sedimentation of cell material of varying diameter, the migration of sedimented cell material through a gradient is characteristic in that its maximum degree of density is no higher than that of the least dense sedimented material. In the course of 25 centrifugation the sedimented cell material moves through the gradient at a largely consistent speed. The measure i of separation is complete when the first cellular particles reach the bottom of the centrifuge tube. This method is suitable for measures of separation of cell materials which essentially vary in diameter; less so with regard to their degree of density.

0* S:23228A ~sl~rrrm~LIIIIP~la~ larsl 12 Procedure Step (c) A treatment which reduces the potential immunogenicity/antigenicity, such as cultivation, UV-radiation, gamma-rays, incubation with antibodies or encapsulation, concerns common preparation procedures of cell material in cultures for the aforementioned forms of application, which are also described in the publications mentioned in this Application. The cryopreservation may be a preferred subsequent step as a measure of preservation. Due to the special purity of the islets obtained, better results are reached after the application of all methods subsequent to the measures of separation, a factor which represents an improvement to the therapeutic and/or diagnostic findings.

The following Example illustrates the invention, without limitation of the latter, (temperatures are given in Celsius). Data as to the location of manufacturers or editors and publishers of publications is limited to locations in the Federal Republic of Germany.

EXAMPLE

1. Pancreatic Tissue The pancreas is removed from a brood sow (German Federal hybrid breeding program, 528 pounds, 34 months) after being shot, bled and relieved of its intestinal 25 tract with cautious preservation of the encapsulated organ from the surrounding fatty tissue, while taking sterile precautions. A 10 minute period of warm ischaemtia is involved in the slaughter process itself.

The thus obtained organ is transported to the islet 30 laboratory in a 4 0 C 39 0 F) cold Eurocollins® solution (Fresenius, Bad Homburg). The time of cold ischaemia prior to beginning with the islet isolation is minutes.

2. Preparation and Distension of the Pancreas All other steps of the islet preparation are carried out under sterile conditions. Only the cut of pancreas closest to the spleen is used for islet isolation. The weight of the prepared organ-segment is 120 grams. The *e.

S..

S S

S

S:23228A r I- 13 pancreatic outJet (ducts pancreaticus) is sought out at the cut between the right thigh and close to the spleen and cannulated with the use of a 19G cannula which is secured with surgical thread.

The weight of the pancreatic cut used from the vicinity of the spleen determines the volume of distension solution. 2 ml of distension solution, i.e. a total of 240 ml per gram of pancreas are injected retrogradely into the ducts pancreaticus through a horizontally positioned cannula. For the distension one may use University of Wisconsin (UW-solution) (DuPont, Bad Homburg) with 0.4% (wt/vol) collagenase (Serva, Heidelberg, catalogue no. 17449), at a temperature of 220C (72 0

F).

After the distension has been completed, more surrounding fatty tissue and connective tissue, the encapsulated organ, and non-distended parenchyme are removed (a total of 15 grams without cannula, i.e. the weight of the pancreas to be used is 105 grams).

20 3. Pancreas Digestion Islets of Langerhans (islets) are isolated according to Ricordi C. et.al: Diabetes 37:413-420, 1988, as well as the method described in the published PCT Application WO 88/09667 for the isolation of human Islets of 25 Langerhans from the combined tissue along with exocrine fragments, ducts elements, vessel fragments, lymph nodes, S' etc.

'For this purpose, the distended pancreas-segment is 0placed in the bottom part of a 450 ml digestion chamber, the structure of which is described in the PCT Application WO 88/09667, along with seven glass balls (marbles). Then the lower portion of the chamber, a 400 g size sifter, the chamber's lid, and a tubing system for the recirculation of the collagen solution are assembled as described in Fig. 2 of the PCT Application WO 88/09667.

The tubing system is filled with additional Hank's solution, the pump is switched on for recirculation of S;23228A Ila~- rr -rrraaPY 14 the solution, and the flow rate is set to 200 ml/min.

The digestion chamber with the distended pancreas and the glass balls (marbles) is subjected to movement in the vertical axle, at a frequency rate of 300 oscillations per minute and an amplitude of 10 mm. The temperature in the digestion chamber is increased with the use of a heat exchanger by 30 per minute, to 32-34 0 C (90-94 0 During the recirculation phase (Phase I) the temperature is maintained constant at this level.

With the aid of microscopic observations of samples taken from the tubing system at 1-2 minute intervals the progress of pancreas digestion may be followed carefully.

After 14 minutes a large number of free islets is first identified in a sample. Recirculation of Hank's solution containing collagenase is stopped at this time, and the chamber flushed with fresh Hank's solution free of enzymes for 15 minutes, at a flow rate of 250 ml/mnin., until there are no more islets visible in the samples (Phase II) The temperature during Phase II decreases 20 from 32-34°C to 150 within 15 minutes. To protect the liberated islets from the influence of collagenase and other proteolytic enzymes released by the pancreas, the tissue suspension is placed in cooled 250 ml centrifuging cups with cone-shaped bottoms, containing 25 35 ml of Hank's solution.

These centrifuging cups filled with tissue suspension undergo 2 minutes of centrifugation at 4°C (39 0 F) in a field of gravity of 120 x g; the rest is disposed of, the complete pellet is filled to 200 ml with UW-solution 4°C and is then resuspended and stored at 8°C (48°F).

Representative samples are taken to determine the number of islets, the islet volume, the DNA content, the insulin concentration and the amylase activity.

4. Isopyknic Density Gradient Centrifugation After another centrifugation (2 min. at 4 0 C in a field of gravity of 120 x g) the rest is disposed of, the concentrate (pellet) re-suspended with a volume of 32 ml S:23228A I st s I ~s~BB IIII~C~- -U 1 15 in 15 ml heat-inactivated newborn calf serum (NCS, Biochrom, Berlin) and transferred into a 600 ml glass cup which is filled up with 400 ml Ficoll-sodium-diatrizoatesolution. This solution is produced by mixing 500 ml Seromed® -Ficoll Separator (1.090 grams per cubic centimetre, Biochrom, Berlin), 12 ml HEPES 1M (Gibco, Eggenstein), 5 ml Penicillin-streptomycin (10.000 IU/ml 10.000 Ag/ml Flow, Irvine, GB), 1 ml 1 N NaOH and ml Medium 199 (Ix, Biochrom, Berlin). The contents of the glass cup (bottom layer) is transferred to a 500 ml blood transport bag (Biopack Model, Biotrans, Dreieich), which is connected to a disposable system previously installed into the blood cell purification device 2991 (Cobe, Kirchheim/Munich) (Blood Cell Processor Processing Set, Cobe, Catalogue No. 912-647-819), in accordance with regulations. The contents of the blood transport bag are then filled into the disposable system. The Blood Cell Processor's Centrifuge is accelerated to 2000 RPM for a period of 2 minutes. 200 ml Medium 199 are layered on to 20 the bottom layer with the aid of a hose pump at a 60 ml per minute flow rate, while the centrifuge is accelerated to 2400 RPM. The disposable system is subsequently **go de-aerated and the supply line is closed. After 10 minutes of centrifugation at 2400 RPM at room 25 temperature, and at the same RPM rate with an integrated "gradient unloader" the following fractions are transferred from the "processing set" to the centrifuging cup in subsequent order: Fraction I: 180 ml Fraction IIa: 25 ml .Fraction IIb: 10 ml Fraction IIc: 10 ml Fraction IId: 10 ml Fraction IIe: 25 ml Fraction III: 290 ml After turning off the centrifuge the "processing set" is taken out and Fraction IV: 50 ml S:23228A 16 is transferred to a glass cup.

The fractions are suspended in Medium 199 with NCS and are centrifuged at 250 x g at room temperature for 5 minutes. The rest is disposed of, the pellet of fractions is resuspended separately in Leibovitz Medium (Biochrom, Berlin) with 10% fetal calf serum (FCS, Biochrom, Berlin), 1% penicillin-streptomycin solution and 2mM glucyl/glutamine (Biochrom, Berlin); the suspensions are transferred to culture vials and evaluated with regard to their islet purity (percentile of share of surface covered with islet tissue).

The fractions containing the mass of islet volume are nearly without exocrine contamination and are subsequently placed into isokinetic gradient centrifugation.

Representative samples are taken to determine the number of islets, the islet volume, the DNA content, the insulin concentration, the amylase activity, the glucose-dependent insulin secretion in-vitro, as well as 20 immunogenicity.

5. Isokinetic Gradient Centrifugation In 100 ml round-bottom centrifuging tubes made of 0 polycarbonate of 16 mm height and 31.5 mm diameter c.

(Nalge, Braunschweig) a bottom layer (cushion) consisting i" 25 of FICOLL and Leibovitz Medium of 4 ml volume, 15 mm height, 1.026 grams per cubic centimetre density and a viscosity of 3.5 cP at 16 0 C is pipetted.

With the aid of a dual-chamber gradient-former, a continual gradient of 76 ml volume and 120mm height, a density rate of 1,026 to 1,017 grams per cubic centimetre and a viscosity range of 3.5 cP to 2,0 cP at 16°C o "(60 0 consisting of FICOLL and Leibovitz Medium, is added on to the bottom layer (the cushion).

Onto this continual gradient we add a 5 ml sample corresponding to 8 mm height and up to 50,000 islet equivalents (one islet equivalent =volume-corrected islet number, an islet equivalent represents an islet of 150 ym diameter, see Ricordi C. et al. Acta Diabetol Lat 27: S:23223A -rr II ~pl~RIFI-"C~~ ~SCSP PPIIBIPlg 17 185-195, 1990) and a remaining cellular mass in Leibovitz Medium.

The centrifuging tube with bottom layer (cushion), gradients and sample are positioned in a centrifuge in such a manner that the distance of the layer between cushion and gradient is 22.2 cm, and the distance of the layer between sample and gradient is 10.2 cm from the centre of the Hettich Rotixa RP centrifuge (Hettich, Tuttlingen) having a 5094 type pivotal rotor and suitable fittings and reductions to contain 100 ml centrifuging tubes. Other centrifuging tubes of the same content or balance tubes are positioned at the opposite end.

The centrifuging tubes are accelerated to 490 RPM for 30 seconds. At this RPM rate a centrifugal force of 27.4 x g acts upon the layer between sample and gradients, and of 59.6 x g upon the layer between bottom layer and gradient. Centrifuging is performed for seconds at 160, slowing to 0 RPM over 30 seconds.

The centrifuging tube is taken from the centrifuge; the first fraction consisting of 80 ml and the second fraction consisting of 5 ml diluted with Leibovitz Medium are aspirated, with another centrifugation at 120 x g, at rso 160 over a period of 2 minutes. The rest is disposed of the remaining cell suspension is resuspended wt.th Leibovitz Medium or other culture media and conventional additives.

Representative samples are taken to determine the *number of islets, islet volume, the DNA content, insulin concentration of the amylase activity, the 30 glucose-dependent insulin secretion in-vitro, as well as immunogenicity.

9 o The purity of the preparation of the isolated Islets ":of Langerhans according to the present invention may be clearly recognised under a light microscope.

The islet suspension obtained is made available for further applications, such as culturing, for example.

6. Examination The islet number, the islet volume, the insulin S:23228A ~e ~e L-l ~dl 18 concentration, the micro-fluorometric viability test, the glucose-dependent insulin secretion in-vitro are determined by their individual samples according to International Standards (Ricordi C. et al. Acta Diabetol.

Lat., loc. cit.). Determination of the DNA content occurs in accordance with the method by Labarca C. and Paigen Anal Biochem 102: 344-352, 1980. The amylase activity of the individual samples is determined with the aid of an a-amylase EPS® Kit (Boehringer, Mannheim, Mannheim). To determine immunogenicity a mixed lymphocyte-islet-culture is performed, see Ulrichs K. et al: Horm. Metabol. Res. 25/S: 123-127, 1990. 100 islets of pig are cultured together with 10 to the power human lymphocytes at 370C (99 0 F) for three days; subsequently, after adding 10 Ci 3H-thymidine one co-cultivates islets and xenogenic lymphocytes for a period of 20 hours. Lymphocyte proliferation correlates to the 3H-thymidine addition and is expressed through an -counter in terms of "counts per minute", under 20 consideration of the control values after conversion expressed as stimulation index.

ee o e S:23228A =I

S~

S S S 19 Table 1 Results of a Preparation of Islets of Langerhans from the Pancreatic two subsequently performed cleaning procedures (isopyknic centrifugation).

Tissue of Pig, using isokinetic gradient Islet number Islet volume (pl) Amylase (U) Insulin (U) DNA (mg) Viability Perfusion Immunogenicity (Stim. Index) Prior to IPGZ 219,985 623 729,644,76 379.6 365,508 After IPGZ 156,849 458 402,773 236.3 6,643 98.7 PI: 0.48 28.5 Prior to IKGZ 152,332 445 165,916 212.6 2,321 After IKGZ 99,016 432 72,748 151.5 1,820 98.5 PI: 4.44 2.3 0 in Islet number Islet volume Amylase Insulin

SNA

0.50% Prior to IPGZ 100% 100% 100% 100% After 2PGZ 71.3% 73.5% 0.055% 62.2% Prior to IKGZ 69.2% 71.4% 0.023% 56.9% After IKGZ 45.0% 69.3% 0.010% 39.9% 82%0.63% 100% S:23228A 20 Table 2 Results of further islet preparations from pancreatic tissue of pig with two subsequently performed cleaning procedures (isopyknic isokinetic gradient centrifugation) Medium values SEM after IPGZ after IKGZ Islet volume (il) 321 44.2 Viability 99.2 0.3 Immunogenicity 100%* (stimulation index after IPGZ= 100%) 315.5 39.4 99.1 0.4 24.4% 6.4%* ii o i ii o i i r ii i iii i ii iii i i ii ii ii ii ii iii ii i i i i i ii i IPGZ: isopyknic centrifugation IKGZ: isokinetic centrifugation PI: perfusion index *Variations in stimulation indications highly significant (p 0.001) S:23228A c.

Claims (34)

1. A preparation of isolated Islets of Langerhans having a purity level of mora than 98%, said purity level being relative to the combined volume of the Islets of Langerhans and other ceil material in the preparation.

2. A procedure for the producing a preparation of isolated Islets of Langerhans of a purity greater than 98% relative to the volume of purified cellular material, comprising: a) distending pancreatic tissue with a physiologically acceptable liquid and extracting Islets of Langerhans with additional cellular material out of the pancreatic tissue; and b) separating the extracted Islets of Langerhans from said additional cellular material by sedimentation based on variation in densities between the Islets of Langerhans and the additional cellular material; and separating the extracted Islets of Langerhans from said additional cellular material by sedimentation based S 20 on variation in diameters between the Islets of Langerhans and the additional cellular material. *fee

3. A procedure for the separation of a plurality of Islets of Langerhans from additional cellular material, o the additional cellular material comprising exocrine, 25 ductal, vascular and lymphoid elements, comprising the steps of: distending a pancreas with a physiologically acceptable liquid; extracting the plurality of Islets of Langerhans and 30 the additional cellular material through digestion of the S.pancreas; separating the extracted plurality of Islets of Langerhans from said additional cellular material by sedimentation based on variation in densities between the Islets of Langerhans and the additional cellular material; and separating the extracted Islets of Langerhans from said additional cellular material by sedimentation based S:23228A 1 I s 22 on variation in diameters between the Islets if Langerhans and the additional cellular material.

4. A procedure according to claim 2 or 3, wherein the physiologically acceptable liquid contains collagenase and/or other proteases.

A procedure according to claim 4, wherein the physiologically acceptable liquid contains collagenase.

6. A procedure according to claim 2 or 3, wherein the Islets of Langerhans and additional cellular material are extracted from the distended pancreas by enzymatic or mechanical influence.

7. A procedure according to any one of claims 2 to 6, wherein said sedimentation based upon varying diameters of cell material is isokinetic gradient centrifugation.

8. A procedure according to claim 7, wherein the isokinetic gradient centrifugation is carried out in a physiologically acceptable aqueous medium with a line.r density gradient. 20

9. A procedure according to claim 8, wherein the aqueous medium contains polysucrose having a molecular weight of approximately 400,000.

10. A procedure according to claim 7, wherein the sedimentation based on varying diameters involves the step of providing in a suitable container a bottom layer consisting of colloid and a physiologically acceptable aqueous medium, followed by another layer of medium having a continual gradient with a density range of approximately 1.005 to 1.600 grams/cm 3 and a viscosity 30 range of approximately 1.1 to 15 cP at approximately to 37°C.

11. A procedure according to any one of claims 2 to wherein the sedimentation based upon varying densities is isopyknic gradient centrifugation.

12. A procedure according to claim 11, wherein the isopyknic gradient centrifugation is carried out in an aqueous medium with discontinuous density gradients.

13. A procedure according to claim 12, wherein the S:23228A g C ACIIIILIPPII~I~I---- 23 aqueous medium contains polysucrose with a molecular weight of approximately 400,000 and sodium-diatrizoate.

14. A procedure according to any one of claims 2 to 13, further comprising the step of subjecting the isolated Islets of Langerhans' to a treatment to reduce their potential immunogenicity/antigenicity, and/or preserve the isolated islets.

A procedure according to claim 14, wherein the treatment to reduce the potential immunogenicity/ antigenicity of the isolated Islets of Langerhans is selected from the group consisting of culturing, UV-radiation, gamma-radiation, incubation with antibodies or encapsulation.

16. A procedure according to claim 14, wherein the preservation of the isolated Islets of Langerhans comprises preserving the islets in culture and/or by cryo-preservation.

17. A procedure according to any one of claims 2 to 16, wherein the step of separating the extracted Islets 20 of Langerhans by sedimentation based on variation in diameters is performed after the step of separating the extracted Islets of Langerhans by sedimentation based on *S* variation in densities.

18. A preparation of Islets of Langerhans prepared by a procedure as defined in any one of claims 2 to 17.

19. A preparation of Islets of Langerhans according to claim 18, wherein diameters of a majority of the isolated Islets of Langerhans are greater than 100pm.

A preparation of Islets of Langerhans according 30 to claim 18 or 19, wherein the purified Islets of Langerhans have a loss of less than 3% islet volume compared to that of the Islets of Langerhans when prepared by said sedimentation based upon varying densities and prior to said sedimentation based upon varying diameters.

21. A preparation of Islets of Langerhans according to any one of claims 18 to 20, wherein viability of the purified Islets of Langerhans is substantially not S:23228A -I 24 o a a a a. 0004 *005 'a a a. adversely affected compared to that of the Islets of Langerhans when prepared by said sedimentation based upon cellular densities and prior to said sedimentation based upon varying diameters.

22. A preparation of Islets of Langerhans according to any one of claims 18 to 21, wherein secretion of insulin by the Islets of Langerhans is substantially not adversely affected compared to that of the Islets of Langerhans when prepared by said sedimentation based upon cellular densities and prior to said sedimentation based on varying diameters.

23. A preparation of Islets of Langerhans according to claim 18, wherein the preparation has a reduction in immunogenicity of about 75% compared to a level of immunogenicity of a preparation of the Islets of Langerhans when prepared by said sedimentation based upon varying densities and prior to said sedimentation based upon varying diameters.

24. A preparation of Islets of Langerhans according 20 to claim 18, wherein the purified Islets of Langerhans have an increase in recovery of islet volume after one or more immuno-modulated treatments compared to the Islets of Langerhans when prepared by said sedimentation based upon varying densities and prior to said sedimentation based upon varying diameters.

A preparation of Islets of Langerhans according to claim 18, wherein the purified Islets of Langerhans have an amylase content lower than 0.05% compared to the Islets of Langerhans following extraction from the 30 distended pancreatic tissue but prior to any said sedimentation step.

26. A preparation according to claim 1 or any one of claims 18 to 25, wherein the isolated Islets of Langerhans are of human origin.

27. A preparation according to claim 1 or any one of claims 18 to 25, wherein the isolated Islets of Langerhans are of animal origin.

28. A preparation according to claim 27, wherein the S:23228A Se~A 25 isolated Islets of Langerhans are derived from pig pancreatic tissue.

29. A preparation according to claim 1 or any one of claims 18 to 28 in the form of an injectable suspension.

30. A method of treating a mammal for diabetes mellitus, comprising the step of administering a preparation of Islets of Langerhans as defined in claim 1 or any one of claims 18 to 29 to the mammal.

31. A method according to claim 30, wherein the mammal is a human being.

32. A method according to claim 30 or 31, wherein said administering comprises injecting the preparation into the vena portae, the vena lienalis, the spleen pulpa, beneath the renal capsule, into an omentum flap, or into an epiploic flap of the mammal by intra- peritoneal, intra-muscular, subcutaneous, intracerebral or intratesticular injection.

33. A preparation of isolated Islets of Langerhans with the purity level of more than 98% substantially as S 20 herein described with reference to the Example.

34. A procedure for the producing of a preparation of isolated Islets of Langerhans of a purity level of 98% i" substantially as herein described with reference to the Example. 25 Dated this 26th day of March 1997 BERNHARD J HERING By their Patent Attorneys GRIFFITH -ACK o o• S23228A Iy INTERNATIONAL SEARCH REPrCT I-t tonal A International Application No. PCT/EP 93/02178 A. CLASSIFICATION OF SUBJECT MATTER JPC 5 A61K35/39 Accordmin to International Patent Classiicaton (IPC) or to both national classification and IPC B. FIELDS SEARCHED II Minimum documen.tion searched (classification system fotlowed by classificaton symbols) IPC 5 A61K Dorumcntiaon searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the internatonal search (name of data base and, where practical, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category' 'itation of document, with indicaton, where appropnate, of the relevant passages Relevant to claim No. Y BIOLOGICAL ABSTRACTS vol. 92 1,3, 1991, Philadelphia, PA, US; 8-11, abstract no. 98540, 15-18, MARCHETTI, PIERO ET AL. 'AUTOMATED 21-23 LARGE-SCALE ISOLATION IN VITRO FUNCTION AND XENOTRANSPLANTATION OF PORCINE ISLETS OF LANGERHANS' see abstract TRANSPLANTATION vol. 52, no. 2 1991 pages 209 213 Y EP,A,O 191 613 (WASHINGTON UNIVERSITY) 20 1-3, August 1986 8-11, 15-18, 21-23 see page 25, line 4 page 27, line 4 Further documents are listed in the continuation of box C. Patent family members are listed in annex. SSpecial categones of atcd documents Speal categories of ted documentsT later document published after the international filing date or priority date and not in conflict with the application but A' document defining the general state of dhe art which is not cted to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international .X document of particular relevance; the claimed invention filing dat cannot be considered novel or cannot be considered to document which may throw doubts on ononty claim(s) or involve an inventive step when the document is taken alone which is cted to establish the publication date of another document of particular relevance; the claimed invention ctation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu. other means mrnts, such combination being obvious to a person skilled document published pnor to the international filing date but in the art. later than the priority date claimed document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report 11 November 1993 07 12. 93 :;ame and mailing address of the ISA Authorized officer European Patent Office, P.B ,l 8 Patentlaan 2 NL 2280 HV Rijswi]k Tel. (i 31-70) 340-2040, Tx. 31 651 ep nl, REMPP, G Fax (+31-70) 340-3016 Form PCT/ISA/210 (second heet) (Jul, 19%? page 1 of 2 -Ld U 41-s 1 INTERNATIONAL SEARCH- REPORT International Application No. PCT/EP 93/02178 C.(Conanuation) DOCUMENTS CONSIDERED TO BE RELEVANT- Cateory itaion of document, with indication, where appropriate, of the relevant passages Relevant to claim No. DIABETES vol. 38, no. SUPi January 1989 pages 136 139 GARTH L. WARNOCK ET AL. 'VIABLE PURIFIED ISLETS OF LANGERHANS FROM COLLAGENASE-PERFUSED HUMAN PANCREAS' DAS GANZE ARTIKEL BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 79, no. 3 1977 DULUTH, MINNESOTA us pages 823 828 ANTONIO BUITRAGO, ET AL. 'RAPID ISOLATION OF PANCREATIC ISLETS FROM COLLAGENASE DIGESTED PANCREAS BY SEDIMENTATION THRO11GH PERCOLL TM AT UNIT GRAVITY' DAS GANZE ARTIKEL 1,2, 8-11, 15-18, 21-23 1-3p 8-11, 15-18, 21-23 loffn PCTIlSA/210 (continuation of second sheet) (July 1992) page 2 of 2 INTERNATIONAL SEARCH REPORT lnionnauon on patent, famiuly mt~bcrs nternational Applicationi No, PCT/EP 93/02178 Patent document I Publication PtnfaiyPublication cited in search report :E date mbes)dale EP-A-0191613 20-08-86 US-A- 4868121 19-09-89 JP-A- 61183226 15-08-86 Form PCT/ISA111 (patet family annex) (July 1992) INTERNATIONALER RCHL3-RCK6NJ3ER1C1WT InternAti 10(c Aencelchcn PCT/EP 93/02178 A. KLASSIFIZIERU NG DES ANM ELDUNGSGEGENSTANDES IPK 5 A61K35/39 Nach der fittntuatonalen Patentassifikation (113K) oder nach der nationallen Klassiflkation und der 1PK B. RECHERCHIERTE GEBIETE Recherchicrter Mindestpniitstoff (K asifikationssystern und Klamfikatianssymbole) IPK 5 A61K( Recherchierte aber rucht zum Mindcstpnifittoff gehobrende Verdffentlichungeri. soweit diese unter die recherchicrten Gebiete fallen Wihred der internationalen Recherche konstitiertc clcktrorusche Datenbank (Nitzne der Datenbank und evii. verwendete Suchbegnffe) C. ALS WESENTLICH ANGESEH-ENE UNTERLAGEN Kategon;:' Bezcichnung der Veraifentlichung, soweit erforderlicb unter Angabe der in B3etacht kornxcndcn Tale Bctr. Anspruch Nr, Y BIOLOGICAL ABSTRACTS vol. 92 1,3, 1991, Philadelphia, PA, US; 8-11, abstract no. 98540, 15-18, MARCHETTI, PIERO ET AL. 'AUTOMATED 21-23 LARGE-SCALE ISOLATION, IN VITRO FUNCTION AND XENOTRANSPLANTATION OF PORCINE ISLETS OF LANGERHANS' siehe Zusammenfassung TRANSPLANTATION Bd. 52, Nr. 2 1991 Seiten 209 213 Y EP,A,O 191 613 (WASHINGTON UNIVERSITY) 20. 3 August 1986 8-AI, 15-18, 21-23 siehe Seite 25, Zeile 4 Seite 27, Zeile liiWeitere Verolfentlichungen sind der Fortsmiung von Feld C zu J Siehe Anhang aetmli *Besondere Kategonen von angegebenien Ver6ffentlichungen Spitere Ver~ffentlichung, die liach demn internationalen Annededatum Veraffentlichung, die den algpemen Stand der Technik definiert, oder dem Priontitsdatum ver~iffentlicht warden ist und mit der abet nicht als besonders bedeulsamn anztzsecen is Arineldung nieht kcillidiert. soadern nur zurnVerstaindnis des der W ilteres Dalcument, das jedocb erst am ader nach dem internaticnalen Teridngzure llge den istnisoerdrzrzgrnei ne Annieldedatum veroffentlicht worden ist er6ffentlichung von besonderer Bedeutung-, die beanspniehte Erfmidung VL Ver6ffentlichung, die geeignet ist, cinen Priaritftsanspruch zweitelhaft er- kann allein aufgrud dieser Ver~ffendichung nicht als neti odcr auf schien zu lassen, oder durch die das Ver~flendichunsdatuna citer erlindenischer Titigecit berithend barachtet werden anderen im Recberchenbericht gcnaninten Ver~lentiichung belcgt werden Veri~frentlichung von besonderer fleceutunig die beanspniehte ErfindunE soil ader die aus elnem anderen besonderen Grund angegebcn ist (wie kann nieht als auf erfinderischer TUdgkeit beruhend betrachtet ausgeflihrt) werden, wenin die /e .niehj mit etner oder mehreren anderen Veroffentliehung, die sich auf eine miindliche Offenbarunig, Ver~fentlichungen dieter Kategonmt in Verbindung gebraclfl wird und tone Benutzunig, etne Ausstellung oder andere Ma~rnahnen beziehit diese Verbindung ffr cimen Facbm~nn naheliegend itt Ap eofnhhn, die var dem intemnationalen Anmeldedatum. abet nach WVretihnteM~e es e aetaii s dem beanspru lten Priontitsdatumn ver~ffentlicht warden istedfnlcug deM~le esienPtnfnii e Datum des Abschlusses der internationalen Recherche Absendedatum des internationalen Richerchenbeuichts 11. November 1993 7 12 Name und Postanschnift der Internationale Recherchenbehorde Bevollmnichtigter Bedicnsteter Euroipisehes Patentarrit, P.B. sai a Patentiaan 2 NL 2280 HV Rijswijk Tel. 31-70) 340.2040, Tx. 31 651 epa W, RM P Faxc 31.70) 340-3016 RM P Fonnbiatt PCT/ISA/31 0 (Biatt 2) (Juli 1993) Seite 1 von 2 INTERNATIONALER RECIHERCHENBERIGHT IntcMA0Q,q0c 6kkzichen PCT/LP 93/02178 C(Fortsmtung) ALS WESENTLICH ANGESEHENE UNTERLAGEN Katcgone' Bezeichnung dcr Vcrbffentlichung, sowat erforderlich linter Angabc der in Elctracht konimenden Talc Bctr. Anspruch Nr. DIABETES Bd. 38, Nr. SUPi Januar 1989 Seiten 136 139 GARTH L. WARNOCK ET AL. 'VIABLE PURIFIED ISLETS OF LANGERHANS FROM COLLAGENASE-PERFUSED HUMAN PANCREAS' DAS GANZE ART IKEL BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. Bd. 79, Nr. 3 1977 DULUTH, MINNESOTA us Seiten 823 828 ANTONIO BUITRAGO, ET AL. 'RAPID ISOLATION OF PANCREATIC ISLETS FROM COLLAGENASE DIGESTED PANCREAS BY SEDIMENTATION THROUGH PERCOLL TM AT UNIT GRAVITY' DAS GANZE ARTIKEL 1,2, 8-11, 15-18, 21-23 1-3, 8-11, 15-18, 21-23 r Formblatt PCTIISA'210 (Fomaetung vn Blat2) (bill 1992) Seite 2 Yon 2 oI. NTERNATJ ONA LE3R REC14EJRCI-IEN13ERICGHT Angabcn zu Vcraffntiichungen, die zur sclben Pat~nqvmbic~ch~rcn IntemrAl i,4ca Aktenzeichon PCT/EP 93/02178 Im Recherchenbericht I Datum de Mitglied(er) der I Datum der angefilhrtes Patentdokument VerbffentlihunS Patirntfamilic EP-A-0191613 20-08-86 US-A- 4868121 19-09-89 JP-A- 61183226 15-08-86 'rmblatt PCT/19A1210 (Anhang Psientfamie)(Jull 1992)