Use of bismuth-213 conjugates for the treatment of micrometastasis

Abstract: The present invention relates to the field of site directed therapy. More specifically it relates to site directed radio therapy. It provides a method for production of radioimmuno conjugates and an apparatus for radioimmuno therapy. The method, conjugates and apparatus can be practicalized without the need for radioactive shielding and/or airtight facilities. Without these restrictions the invention provides a simple and efficient means of therapy at the bed-side of the patient.

Description: The present invention relates to the field of site directed therapy. Nowadays there are a number of methods of site directed therapy which have been suggested to eliminate unwanted cells or infectious organisms from the body of a mammalian subject. There are many fields of therapy in which said methods may be applied. The most important ones seem to be immune diseases (either auto immune diseases or acquired immune diseases), cancer and viral or microbial infections. Site directed therapy is a method whereby a cytotoxic compound is delivered to the immediate vicinity of the target cell or infectious organism. This is usually done by coupling a targeting moiety to the cytotoxic compound. This targeting moiety recognizes a structure in, on, or near the target. Known targeting moieties include, but are not limited to, antibodies, more specifically monoclonal antibodies and more preferably human monoclonal antibodies, nucleic acids, receptor directed ligands and the like. Cytotoxic compounds can be for instance drugs, such as adriamycin, toxins such as ricin A and radioisotopes. Radioisotopes cannot only be used for therapy, but they can also be used to identify the site or sites of the target (imaging). This invention provides methods of therapy and imaging using a conjugate of a targeting moiety and at least one radioisotope. Therapy with targeting moieties is widely known. Targeting can be accomplished by aiming the targeting moiety directly to the wanted site, but it may also be directed to another targeting moiety which is directed to the wanted site (so called pretargeting). Pretargeting offers an advantage over direct targeting when the specificity of the targeting moieties is not sufficient. By using a first localizing moiety followed by a second one coupled to a cytotoxic compound, the amount of cytotoxic compound delivered to non-target sites can be lowered significantly. Known targeting moieties, such as antibodies, often cannot be provided with a large amount of cytotoxic compounds without hampering their targeting specificity. Therefore it has often been suggested to use a carrier molecule, such as HSA or a nucleic acid, or a polymer, which can be loaded with a high number of cytotoxic compounds and coupled to a targeting moiety. All of the above-mentioned variations on the theme of site directed therapy and/or imaging can be used more advantageously with the present invention. A well established problem in the field of imaging and site directed radiotherapy is to find a suitable radioisotope. Apart from the amount of energy that is released upon their decay, which should be sufficient to be measurable outside the subject in the case of imaging and sufficiently lethal to the target in the case of therapy, there is also a problem in finding an isotope with a suitable half-life. An isotope with a long half life cannot be chosen because of the biological half life of the targeting moiety, which means that most of the isotopes will decay after disintegration of the conjugate. This decay after the disintegration of the conjugate will lead to cytotoxicity to other cells or tissues than the target. Furthermore, all conjugates which do not localize will be secreted from the body and present a radio active waste problem. It is also not practical to choose a radioisotope with too short a half life, because of packing and shipping delays and because the institution carrying out the therapy must be equipped to make the conjugate, transport it to the patient and administer it in a very short interval of time, otherwise most of the radioisotope will have decayed before entering the body, let alone localization at the target site. The isotopes used for imaging usually are gamma emitting isotopes, for therapy auger electron emitting α,β-emitting, or α- emitting isotopes may be used. Most preferred for the present invention are α-emitting isotopes. The short-range cell-killing effect of α-particles is enormous: a 1 mm diameter tumor, comprising maybe 600,000 cancer cells needs about 6 α-particles of 6 MeV per cell to deliver a dose of 600 rad, causing a 99.9 % cell-kill ratio, and that specifically because of the stochastic nature of the hit- and kill-mechanism. However, due to the same stochastic nature, a 10 times lower α-radiaton dose will enhance the cell-survival ratio with a factor 500: more than 50% of the cells (or non-tumor cells in similar morphology for that matter) would survive a 60 rad α-radiation dose, equivalent to 0.6 α-particles per cell. This characteristic would make an effective α-radioimmunotherapy within reach, provided that a "quality factor" for the isotope-antibody conjugate of 10 or better can be obained. It is the purpose of the present invention to contribute towards this goal in a most essential manner. The quality factor is a ratio between localized antibody at the target site, divided by the antibody "sticking" to other tissue. The notion of using α-particles emanating radioisotopes as agents for the killing of tumor cells was already mentioned in the literature during the mid-fifties. Since then other potential candidate-isotopes were and are being proposed, of which a good summary is given by Fisher (1) and by Wilbur (2) which brings the list to (with their half-lifes between parentheses): 223 Ra (11.4 d), 225 Ac (10 d), 224 Ra (3.6 d), 225 Fm (20 h), 211 At (7,2 h), 212 Bi (60 m), and 213 Bi (47 m) . Although important publications appear regularly in the literature regarding microdosimetry, antibody-isotope coupling techniques, pre-clinical in vitro and in vivo experiments, no clearly defined, larger scale clinical experiments are being done until now, for a variety of reasons: a. no human monoclonal antibodies with proven sufficient quality are available yet, b. no biological safety data are available for antibody coupling agent (the latter for the binding of the radioisotope) combinations, c. some isotopes may not become available for large scale application at acceptable costprices ( 225 Fm), d. isotopes may be too difficult and therefore too expensive to obtain because of the necessary procurement process ( 211 At from 209 Bi by a (α, 2n) reaction in a cyclotron and subsequent isolation plus purification), e. other isotopes do have a Rn-isotope as first daughter in their decay sequence, allowing redistribution of daughter nuclei before decay ( 224 Ra, 223 Ra), and also necessitating gas-tight reaction conditions, f. some isotopes may have a relatively long living daughter isotope somewhere in their decay sequence ( 224 Ra, 223 Ra, 225 Ac), also with the chance that daughters thereof may redistribute before decay, g. the radioactive halflife of some isotopes is so long that most of the activity leaves the patient undecayed, resulting in a waste problem ( 223 Ra, 224 Ra, 225 Ac), or h. the halflife of the isotope is so short that most of the isotope decays before reaching its ultimate target ( 212 Bi, 213 Bi), i. sufficient precursor material may not be available to extract at one time the necessary amount of isotope for a single patient treatment ( 212 Bi, 213 Bi), and, j. isotopes producing hard gamma rays in their isotope decay need shielding facilities to prevent radiation hazards to technicians and nursing personnel. One or more of the arguments listed above will make it very difficult, if not impossible for some of the isotopes to ever be used on a large scale for α-radio-immunotherapy, and that in particular if one or more of the others can be used on acceptable technical, logistical and financial conditions. The closest prior art to the present invention (the French patent application FR-A-2 527 928) discloses a method to produce conjugates of an antibody and 212 Bi. However, these conjugates still suffer from the drawbacks mentioned above under e, h, i and j. In WO 9015625 the use of actinium-225 or one of its α-emitting daughter radionucleotides 221 Fr, 217 At, 213 Bi and 213 Po in radioimmunoconjugates is disclosed. The present invention concerns the use of a conjugate of a targeting moiety and the 213 Bi radioisotope for the manufacture of a medicament for the treatment of cancer in micrometastasis. Such 213 Bi radioconjugates can be made or quasi be made at the site of therapy. Due to the decay sequence which results mainly in α- and/or β-radiation it has become possible that no protection against radiation is necessary. This is extremely useful, because due to the absence of gamma radiation it has become possible that the conjugation can be done at or near the bed side without the necessity to apply radiation shielding or isolation of the patient. This is not only preferable from the point of radiation danger but is also gives advantages for the availability of the short-living isotope. This isotope can be prepared in the neighboorhood of the patient allowing for a rapid administration and prevention of loss of therapeutic action caused by the rapid decay of the isotope. In this way it has become possible to use short lived radioisotopes for therapy. An ion exchange column or another appropriate substrate filled with the long lived isotope can be placed at or near the bedside, for instance, where the short-lived isotope can be eluted by washing the substrate with a suitable solution. After elution the short lived isotope is coupled to the targeting moiety and (optionally together with an infusion solution) the conjugate can be administered. This can all be done in a continuous mode with an apparatus according to the invention as shown in fig. 1 or fig. 3, or in an intermittent mode by using ordinary laboratory glassware. Of course it may also be made possible to add the targeting moiety to the eluting solution so that the coupling takes place in the column. This invention primarily addresses the use of the shortest-lived isotope from the list mentioned above, 213 Bi. The invention enables the person skilled in the art to milk this isotope by a continuous or an intermittent extraction-method from one of its precursors, 225 Ac, at the bed-side of the patient, or in the nearestby hospital laboratory facility, to link the 213 Bi in a continuous or an intermittent manner onto the targeting moiety, to either or not mix the conjugate solution with an infusion liquid and to administer this mixture intravenously to the patient - for example as is schematically pictured in figure 1. At first sight this procedure might seem extremely wasteful, because 225 Ac, itself being an α-emitting isotope, produces three potentially therapeutically useful α-particles before yielding the 213 Bi-isotope, as is shown in figure 2. However, the source material for 225 Ac, 229 Th, and thereby also the 225 Ac itself, can be made available at sufficiently low cost to allow it to be used in the proposed manner on economically justifiable terms. The use of 213 Bi is not only preferable from a viewpoint of radiation hazards. It is also preferable because no gaseous isotopes occur in the decay sequence of its precursors. This is advantageous over the use of other isotopes which have a decay with a gaseous isotope which necessitates the handling and reaction environment to be air-tight. Milking, conjugation and administration of 213 Bi are not hampered by the necessity for having air-tight conditions and the reactions can be done under normal conditions. The targeting moiety may preferably be a monoclonal antibody, or a fragment or a derivative thereof. Preferably such an antibody is a human or a humanized antibody to prevent immunologic reactions to the antibody. Non-human antibodies are mostly of murine origin. These, like all other foreign proteins, are highly immunogenic in man. The phenomenon of HAMA, human anti mouse antibodies, is well known in the field and severely limits the use of mouse derived antibodies in diagnostic and especially in therapeutic applications in human beings. A single application of a murine antibody is usually sufficient to mount an immune response that will prevent subsequent applications to be effective. Of course fragments and/or derivatives of the targeting moieties can also be used, as long as they retain a substantial amount of target specificity. Thus, for this invention it should be understood that where a targeting moiety is mentioned one should also consider a fragment or a derivative thereof as part of the invention. Preferably antibodies are directed against tumor associated antigens, such as CEA (Carcino-embryonic antigen) , AFP (alpha-foetoprotein), FHAP (fast homoarginine-sensitive alkaline phosphatase), p97 (melanome specific), and EL-1 (elongation factor 1). Another preferable targeting moeity is formed by a ligand for a cell surface receptor or a fragment or derivative of such a ligand. Examples of such ligands are agonists and/or antagonists of pharmacologically active receptors, but also T cell epitopes which can bind to the T cell receptor are prefered. Numerous patients can be treated with one ion exchange column loaded with isotope. The amount of isotope loaded depends on the number of patients to be treated. The wanted isotope can be eluted from the column intermittently, with suitable intervals depending on the half-lifes in the decay chain. With related tumours or infectious organisms the same targeting moiety (or mix of targeting moieties) may be used for various patients. For unrelated diseases there must be a means for changing the targetting moiety preparation. The coupling of the isotope to the targeting moiety can be done in any suitable way, as long as the targeting specificity of the targeting moiety is not hampered to a substantial amount. Preferably the coupling will be done through one of the now many known chelating agents. As already disclosed, it may be advantageous to couple the isotopes to a carrier, such as HSA, which of course can also be done through chelating agents. The advantage of a carrier is that a large number of radioisotopes can be brought to the target cell. Since it is assumed that several α-particles are necessary for the destruction of one target cell an increase in the number of isotopes in the direct neighbourhood of the target cell is preferable. The simplest way to describe a method and apparatus, for the preparation of 213 Bi conjugates for use in the invention, with reference to figure 1, is as follows: A capillary column contains, by means of example, twice the amount of precursor- 225 Ac needed for a single patient dose of 213 Bi. Example: in a case the patient dose corresponds with 30 mCi (equals 2.10 -9 g) of 213 Bi over a 10 day period, the capillary column (3) will contain 200 µ Ci of 225 Ac (equals 4.10 -9 g). The 225 Ac is present in a 3 + form on a suitable ionexchange substrate. Upon its (continuously occurring) decay it is stripped from the column by a certain overdose of the eluent in flask (1) containing the appropriate targetting moiety capable of binding the isotope. The binding part of the targetting moiety and other chemical equilibrium conditions of the eluent-ionexchange system are chosen such that the 213 Bi, for all practical purposes, quantitatively binds to the targetting moiety. The immediate daughter of 225 Ac, 221 Fr has a radioactive decay halflife of 4.8 minutes. It is this isotope which acts via the very short-lived 217 At as the direct precursor of 113 Bi. In case the 221 Fr is not retained by itself or in the ion exchange substrate, the delaying effect of the 221 Fr-halflife causes the need of a certain period of time between the decay of 225 Ac at and its stripping from the capillary column and the binding of the 213 Bi onto the targetting moieties. The optimum value for such a delay is somewhere between the halflife of the 221 Fr and the halflife of the 213 Bi isotopes. This delay can be effected by the length of tubing between the capillary (3) and the patient (4), if necessary enhanced by an extra length of intermediate tubing, as indicated in figure 1 as (5). The infusion liquid from flask (2) enters the patient, it is mixed with the isotope-containing eluate from column (3), as indicated as junction (6) in figure 1. In order to obtain optimal stripping and conjugation conditions in the capillary column (3), it may be that the composition of the eluent in flask (1) is not optimal (for example its pH-value) for administration to the patient. Presuming that the volume rate of infusion liquid is an order of magnitude higher than of the eluate liquid, this can easily be countered for by a compensating off-balance (buffered) pH-value of the infusion liquid. It is also possible that the binding of the targeting moiety is hampered by the physico-chemical properties of the eluent. Therefore, an other embodiment of the invention is represented in fig. 3 where an eluens is lead from a vessel (7) through an ion exchange column (3) so that a radioisotope is stripped from that column. The eluens containing the isotope is mixed with a liquid from a vessel (1) containing a targeting moiety, so that the isotope is bound to the targeting moiety. The resulting fluid is mixed with infusion liquid from vessel (2) at junction (6) and administered to the patient (4). Optionally the eluens containing the isotope may be lead through an additional length of tubing (5) to correct for the half-life of intermediate daughter isotopes. What the invention enables in terms of the development and the clinical use of α-radioimmuno-therapy, in this case using 213 Bi as the active cell-killing agent is: "single patient kits" in the form of precursor with a halflife that is logistically managable regarding: minimization of active material loss by radioactive decay during operations like packaging, transport, etc., safety in transportation over long distances and in handling in hospitals, applicability in practice on a large scale in many hospitals without need for special precautions, regarding: the handling of the material and the application procedures regarding the treatment of patients, all without complicated monitoring equipment, collection and handling facilities for (urinous) waste, maximal (and in case of continous extraction,almost total) use of the 213 Bi after it is generated from the precursor isotope, maximum flexibility in dose administration by the possibility of changing treatment time, allowing for a minimum range of single patient kit precursor concentration standards. All these aspects then pertain precisely to fields where the short-range α-particles are most suited for their potential therapeutic uses like: micrometastases (of less than 1 mm diameter) of various cancers, A special advantage of intermittent administration of the therapeutic radioconjugates is the advantage which occurs by dose fractionation. Statistically it is possible to calculate the dose needed to kill 99.9% of the tumor cells with a dose of radioconjugate: assuming that a leukemic (monocellular, blood and marrow bone) tumor load of 1 kg exisits, which is roughly equal to 10 12 cells, and that 10 α-particles are needed to kill a cell (6 MeV), then 10 13 α-particles would be needed, which corresponds with 50mCi 213 Bi. Thus for a single dose, which would kill 99.9% of the tumor cells 50mCi 213 Bi would be needed. The "dose versus survival" relation for this cell morphology with 6 MeV α-particles can be derived from the formula D/D 0 = -In S, in which S = survival fraction, D = dose administered and D 0 = reference dose for 37% survival. From this formula the following Table of values can be calculated: Dose versus kill ratio for tumor cells. The numbers are the number of α-particles necessary to kill the given % of tumor cells. In case A 600 rad are necessary to obtain a 99% kill ratio. In case B 2000 rad is assumed necessary for the same effect. Cell kill in % Case A Case B 0 0 0 1 0.015 0.05 10 0.15 0.5 40 0.7 2 50 1 3 60 1.3 4 70 1.5 5 90 3 10 99 6 20 99.9 9 30 99.99 12 40 99.999 15 50 From this Table the effects of an intermittent, dose fractioned, administration can be read: The effect of cell survival of successive doses of 5 mCi 213 Bi in case A is as follows: the first dose of 5 mCi equals 1 α-particle per cell, which gives 50% survival, which means that 0.5·10 12 cells remain; the second dose of 5 mCi equals 2 α-particles per cell, which gives 20% survival, which means that 0.1·10 12 cells remain; the third dose of 5 mCi equals 10 α-particles per cell, which gives 0.1 % survival, which means that 0.1·10 9 cells remain; the fourth dose of 5 mCi equals 10,000 α-particles per cell, which means a total kill. Thus it can be shown that by intermittent dosing a total dose of 4 times 5 = 20 mCi 213 Bi is sufficient to give a total kill of the tumor cells. For clarity the effects of intermediate tumor growth and maximization of the number of targeting moieties on the tumor cells have been omitted. Nevertheless, it is clear that by intermittent administration the total load of radioactive material can be kept smaller. Even in case B, which has a more unfavourable dose versus survival rate, and advantageous effect is realized: 1 st dose → 1 α/cell → 75% survival → 0.75·10 12 cells 2 nd dose →1.3 α/cell → 70% survival → 0.50·10 12 cells 3 rd dose → 2 α/cell → 60% survival → 0.30·10 12 cells 4 th dose → 3 α/cell → 50% survival → 0.15·10 12 cells 5 th dose → 6 α/cell → 25% survival → 0.04·10 12 cells 6 th dose → 25 α/cell → 0.3% survival → 0.1·10 9 cells 7 th dose → 10,000 α/cell → total kill after 35 mCi. There are two ways presently known to obtain 229 Th as a precursor for the 225 Ac-source-isotope: from stockpiled 233 U, by its natural α-decay. Batches of 233 U were made in nuclear breeder reactors about 30 years ago, but never used as nuclear fuel. Some of the 233 U was separated from the bulk- 233 Th, from which it was made, so that the now available 229 Th can be obtained in highly pure form. by high neutron flux irradiation from natural 226 Ra, with 227 Ac as an intermediate product. Futher irradiation of this 227 Ac yields rdughly equal amounts of 229 Th and 228 Th, the latter with much shorter halflife (2 years) than the 229 Th. On the one hand this complicates the extraction of 225 Ac considerably, but in properly equipped installations it may on the other hand yield 224 Ra, an α-emitter with a 3.7 day halflife. Examples Example 1. The separation chemistry of the various radioactive elements mentioned in the text before has been sorted out decades ago and is well-documented in the public literature. Examples are references (3) and (4). 225 Ac can be separated from 229 Th on a Dowex 50 ionexchanger by stripping with 4N HNO 3 . After evaporation of the acid, the 225 Ac can be dissolved again in 0.5N HNO 3 in a fixed concentration and absorbed in the appropriate amount on Dowex 50, which then becomes the material in the mini-column (3) of figure 3. Example 2. 0.68±0.07 mCi of 225 Ac was obtained from the European Joint Research Centre. This was loaded on a MP-50 cation exchange resin (Bio-Rad). The formed 213 Bi was eluted with a mixture of 50:50 10% NH 4 Ac:MeOH with a pH of 6.75. An autoburet was used to deliver 35 µ l of eluant per minute; alternatively, manual elution was done at 50 µ l amounts of eluant per minute. In a few experiments it was necessary to purify the 213 Bi. This was accomplished by heating the eluant to dryness in a 10 ml beaker containing 0.5 ml of conc. HNO 3 . After evaporation under an IR lamp, the bismuth activity was transferred to a column of MP-50 resin (2x30 cm, pre-equilibrated with 0.1M HNO 3 ). The resin was washed with 0.2 ml H 2 O. Then the 213 Bi was eluted with 0.5 ml of HCl and HI. Various concentrations of HCl and HI have been tried. Figure 4 shows the elution patterns for 213 Bi. In all cases the elution is rapid and quantitative. All of the isotope can be obtained within 5 to 10 minutes after the start of the elution. Example 3. Radiolabeling was done by adding enough 3M NH 4 Ac to the 213 Bi stock to achieve pH 4.0-5.0. Then 53 µ l or 106 µ l of a 4.7 mg/ml solution of monoclonal antibody B3 coupled with the chelator CHX-DTPA (cyclo-hexyldiethylenetriaminepenta acetic acid) according to the method described in (5) were gently mixed into the solution. After a fifteen minute reaction time, 1.5 µ l of 0.1M EDTA were added. The solution was transferred to a 1 ml syringe with 0.2 ml wash. The solution was then injected into the HPLC (high pressure liquid chromatography) having a TSK 3000 column. The buffer was 0.02M MES/Cl - (MES= morpholino ethane sulfonic acid), 0.15M NaCl, pH 6.5. Elution of the B3 antibody occurred at 7.5 minutes. The amount of 213 Bi incorporated into the antibody was monitored with an in-line radiochemical detector (Beckman). All activity measurements of 213 Bi were corrected for decay (t ½ = 45.6 min). Results are depicted in Table 2. Activities of 225 Ac, 221 Fr or 217 At were not detectable in any of the 213 Bi elution products. Results of radiolabeling experiments incorporating 213 Bi into mAb B3-CHX-DTPA. Acid Vol. acid µl µg mAb 213 Bi recovered in mAb (%) 2M HCl 210 250 43.4 (37%) 2M HCl 210 500 45.0 (25%) 0.1M HCl 500 500 3.4 ( 7%) 0.1M HI Example 4. In the same way as described in Example 2 and 3, 213 Bi was eluted from 225 Ac and coupled to a targeting moiety. For this experiment a conjugate of monoclonal antibody M195 and the chelator CHX-DTPA was used. Table 3 summarizes the results. Results of radiolabeling experiments incorporating 213 Bi into mAb M195-CHX-DTPA. µg antibody 213 Bi recovered (%) 50 198 (53%) 25 107 (39%) Legend to the figures Figure 1 depicts an apparatus according to the invention. An eluent containing a targeting moiety is lead from a vessel (1) through an ion exchange column (3) wherein radioisotope binds to the targeting moiety. The RC is mixed with infusion liquid from vessel (2) at junction (6) and administrated to the patient (4). Optionally there is additional length of tubing between column (3) and junction (6) to correct for the half lives of intermediate daughter isotopes. Figure 2 depicts the decay chain of 225 Ac. Figure 3 depicts another apparatus according to the invention. An eluens is lead from a vessel (7) through an ion exchange column (3) so that a radioisotope is stripped from that column.The eluens containing the isotope is mixed with a liquid from a vessel (1) containing a targeting moiety, so that the isotope is bound to the targeting moiety. The resulting fluid is mixed with infusion liquid from vessel (2) at junction (6) and administered to the patient (4). Optionally the eluens containing the isotope may be lead through an additional length of tubing (5) to correct for the half-life of intermediate daughter isotopes. Fig. 4 shows the elution behavior of 213 Bi at several HCl and HI concentrations. References: (1) D.R. Fisher: "α-particle emmitters in medicine", proceedings of a symposium held at Loews L'Enfant Plaze Hotel, Washington, DC, September 21 and 22, 1989, pages 194-214, published by the American College of nuclear physicians. (2) D.S. Wilbur: "Potential use of α-emitting radionuclides in the treatment of cancer", Antibody, Immunoconjugates, and Radiopharmaceuticals, volume 4, number 1, 1991, pages 85-97, published by Mary Ann Liebert, Inc. (3) T. Mitsugashira: "Preparation of traces for actinium, thorium, protactinium and uranium", SPEY, Min. Educ. Sci. & Cult., Tokyo, 9, 1984, pages 111-116. (4) S. Suzuki: "Solution chemistry of light actinide elements", Japan-US seminar on Thorium fuel reactors - proceedings, Nara, Japan, 18 - 22 October 1982 (Tokyo, 1985) pages 137-143. (5) Mirzadeh, S., Brechbiel, M.W., Atcher, R.W., Gansow, O.A., Bioconjugate Chem., volume 1, 1990, 59-65.

Claims: A use of a conjugate of a targeting moiety and the 213 Bi radioisotope for the manufacture of a medicament for the treatment of cancer in micrometastasis having a diameter of 1 mm or less. The use according to claim 1, wherein the targeting moiety is a monoclonal antibody or a fragment or a derivative thereof. The use according to claim 2, wherein the monoclonal antibody is a human or humanized antibody.

Inventors: GEERLINGS MAURITS WILLEM, GEERLINGS, MAURITS WILLEM
Applicants: AKZO NOBEL NV, AKZO NOBEL N.V.
Application year: 1993
Main classification: A61K51
IPC codes: A61K51, G21G4, A61K39, G01N33, A61P35, A61N5, A61M5