Patent Application
20120149058
The present invention relates to a process and an apparatus for testing substances for potential toxicity.
During the drug discovery process a variety of tests are performed to avoid investments in further testing or development of substances which are likely to not be able to fulfil regulatory requirements, e.g. such substances may turn out to be toxic or otherwise be not suitable for the development of a drug. One of these test in a very early phase of the drug discovery process is the so-called “Ames test”.
In essence, the Ames test is a test for determining if a substance is mutagenic and is based on the assumption that a substance that is mutagenic (for the bacteria used in the
Ames-test) may also turn out to be carcinogenic. Although, in fact, some substances that cause cancer do not give a positive Ames test (and vice versa), the simplicity and low cost of this test makes it highly recommendable in the process of screening substance for possible carcinogenicity.
The respective bacteria (strains of salmonella) used in the Ames test carry a defective (mutant) gene making them unable to synthesize the amino acid histidine from the ingredients in the culture medium, thus making them unable to grow on a culture medium lacking histidine. However, some types of mutations can be reversed (back mutation), with the gene regaining its function. These revertants are then able to grow on a culture medium lacking histidine.
Many substances are not mutagenic (or carcinogenic) themselves but become converted into mutagens (or carcinogens) as they are metabolized by the body. This is the reason why the Ames test also includes liver enzymes. In case there is bacterial growth on the culture medium lacking histidine without liver enzymes having been added then the substance itself is mutagenic. If no such bacterial growth occurs without liver enzymes having been added but occurs with liver enzymes having been added then the metabolite of this substance is mutagenic. In either case, the substance is not further considered in further testing or in development of a drug. As already mentioned, the Ames test is routinely used in the drug discovery process due to its simplicity, low cost and its high probability that a substance (or its metabolite) which is marked by the Ames test as being cancerogenic is in fact cancerogenic. In addition, in some countries having performed the Ames test is a regulatory requirement that must be complied with in order to later on get the allowance for a drug to enter into the market.
For the reasons mentioned above, the Ames test is a well-established test in the drug discovery process. However, since nowadays very large numbers of substances are synthesized automatically in only small amounts the tests have to be performed in a highly efficient manner and with these small amounts of the substances. Typically these substances are provided in various differently diluted concentrations in micro-well plates (e.g. in micro-well plates having 96 micro-wells). However, the testing of these substances—or at least some essential steps thereof—is still performed manually to a large extent.
There is therefore a need for a process and an apparatus for efficiently and automatically performing the Ames test, and preferably the process and apparatus should be suitable to use standard laboratory equipment such as micro-well plates, Petri-dishes, standard pipettes, etc., so that in view of the large numbers of substances to be tested with different concentrations the efficiency of the Ames test can be substantially increased. Also, only small amounts of the substances to be tested should be necessary.
To achieve this, the present invention suggests a process as is it specified by the features of the independent claim directed to a process. Further embodiments of the process according to the invention are the subject of the dependent process claims.
In accordance with the present invention the process for automatically testing substances for potential carcinogenicity comprises the steps of:
This process is particularly suitable to automatically perform the Ames test and increase the efficiency of performing the test, since the automatically performed Ames test allows to process a large number of substance/salmonella mixtures in a comparatively short time. Also, it is possible to use standard laboratory equipment such as e.g. standard 96 well micro-well plates, in the wells of which only small amounts of the substance/salmonella mixtures to be tested can be provided with the substance being contained in the mixtures in different concentrations. In addition, while not being mandatory it is possible to use standard Petri-dishes in which a disk of a solid culture medium such as Agar is arranged. The spin-coating of the solid culture medium (Agar) in the dishes, which is achieved by first spinning the dishes after the substance/salmonella mixture has been dispensed on the solid culture medium and then stopping the spinning of the dishes, results in a uniform layer of mixture on the solid culture medium. After having closed the dishes with a lid and after a predetermined incubation time counting of the number of salmonella colonies can be performed easily and reliably.
In one variant of the process according to the invention, the step of providing a plurality of substance/salmonella mixtures to be tested in the wells of a micro-well plate comprises:
This variant is advantageous in that the substances to be tested in different concentrations in form of respective substance/salmonella mixtures in different wells preferably of one and the same micro-well plate can be prepared at a location remote from the location where the micro-well plates are provided for the automatic processing. Once an entire micro-well plate has been prepared, it is either forwarded directly to the location where the plates can be automatically processed, or it is stored in an intermediate storage from where it can be automatically forwarded for processing.
In a further embodiment of this variant of the process according to the invention, the step of providing a plurality of substance/salmonella mixtures further comprises adding liver enzymes in a liquid form to the different dilutions of each substance prior to admixing the salmonella strains. This embodiment of the process is advantageous in that in addition to performing the Ames-test for the substances per se (or the respective substance/salmonella mixture) the Ames-test is also performed for potential metabolites of the substances (which are obtained with the aid of the liver enzymes), so that both the substances per se and potential metabolites thereof are automatically subjected to the Ames-test. Alternatively, a buffer liquid can be added instead of the liver enzymes if appropriate, however, in this case it is the substance itself rather than any metabolite thereof which is subjected to the Ames-test.
In a further variant of the process according to the invention, the respective solid culture medium arranged in the respective dish is heated prior to dispensing the mixtures of substance/salmonella and culture medium from the pipettes into the associated dishes. This pre-heating increases the hydrophilicity of the solid culture medium (e.g. Agar) arranged in the respective dish and thus further improves the uniform formation of the layer of the dispensed substance/salmonella mixture. By way of example, the heating of the solid culture medium arranged in the respective dish can be performed using IR-radiation (InfraRed).
In a further variant of the process according to the invention, the step of intaking into the pipettes a desired amount of liquid culture medium and mixing it with the respective intaken substance/salmonella mixtures comprises aspirating air into the pipettes for performing the mixing of the intaken substance/salmonella mixtures with the liquid culture medium. Through the aspiration of air a good mixing of the substance/salmonella mixture with the liquid culture medium (e.g. Soft Agar) is achieved.
As regards the apparatus for the automatic testing of substances for potential carcinogenicity according to the invention, this apparatus comprises:
Such apparatus allows to automatically perform large numbers of Ames-tests in a comparatively short time using standard laboratory equipment such as e.g. standard 96 well micro-well plates, in the wells of which only small amounts of the substances to be tested can be provided in different concentrations with or without liver enzymes. The micro-well plates are provided on a plate support arranged at a well-defined location, so that different 96 well micro-well plates are always provided at the same location. The movable pipetting unit can be automatically moved to the location of the plate support where the micro-well plate with its 96 different wells is provided. The pipetting unit comprises a predetermined number of pipettes and is capable of automatically intaking from a corresponding number of wells a corresponding number of substance/salmonella mixtures or substance/salmonella/liver enzymes mixtures, respectively, and then moves the pipettes towards a corresponding number of containers each containing a liquid culture medium (e.g. Soft Agar). The pipetting unit then aspirates a predetermined amount of liquid culture medium and a mixture with the intaken substance/salmonella mixture or with the intaken substance/salmonella/liver enzymes mixtures is produced. After the intake of culture medium movable pipetting unit moves on to a dish support where a corresponding number of dishes (e.g. Petri-dishes) each containing a disk of a solid culture medium (e.g. Agar) arranged on the bottom of the dish is waiting. The mixtures contained in the pipettes are then dispensed into a respective spinning dish. The centrifugal forces acting on the respective dispensed liquid mixture forces the said mixture towards the upstanding side wall surrounding the bottom of the dish. Spinning is then stopped allowing the liquid mixture to evenly distribute over the solid culture medium so as to form a uniformly distributed film. Once this film has been formed, the closing unit places a lid on the dish and the closed dishes are then transported towards the intermediate storage, where a plurality of such dishes can be stored. Once the intermediate storage is complete, it can be forwarded to an incubator. After a predetermined incubation time counting of the number of salmonella colonies can be easily and reliably performed.
One embodiment of the apparatus according to the invention further comprises heating means which are capable of being positioned above the the respective solid culture medium arranged in the respective dishes on the dish support. As already mentioned above, these heating means can be used to temporarily heat the solid culture medium (e.g. Agar) so as to increase the hydrophilicity thereof. In a particular embodiment of this apparatus, the heating means comprise a number of IR-heaters corresponding to the number of dishes that can be arranged on the dish support at the same time.
In a further embodiment of the apparatus according to the invention, the pipettes of the pipetting unit are arranged at an inclination angle relative to a normal to the plane of the plate support, with adjacently arranged pipettes being arranged at opposite inclinations relative to the normal. This embodiment is particularly advantageous when using standard micro-well plates such as the afore-mentioned 96 well micro-well plates in connection with standard pipette tips, e.g. 5 ml (or even larger) standard pipette tips, which—while having a tip with a very small diameter—conically increase in diameter. Due to the small spatial distance of adjacent wells of such micro-well plates it is then not possible to arrange these standard pipettes normal to the plane of the plate support if the pipette tips are to be introduced in adjacently arranged wells of such micro-well plates since in this case adjacently arranged pipettes would collide. However, in case adjacent standard pipette tips are arranged inclined relative to the normal to the plane of the plate support at opposite inclinations (e.g. one pipette tip is arranged inclined at a positive angle relative to the normal while the adjacent one is inclined at a negative angle relative to the normal, and so on) then it is possible to use standard pipette tips and 96 well micro-well plates and at the same time it is possible to introduce adjacently arranged pipette tips into adjacently arranged wells of the micro-well plate without collision. The angle of inclincation may be in the range of up to 10° relative to the normal, and amounts in particular about 7.5° relative to the normal.
In a further embodiment of the apparatus according to the invention the pipetting unit comprises spacing means for laterally moving the pipettes towards and away from one another. These spacing means allow the pipettes to be arranged very closely relative to one another at the location where they intake the substance/salmonella mixtures or substance/salmonella/liver enzymes mixtures from adjacent wells of the micro-well plate, and to then move them away from one another so that they can subsequently be introduced into the containers containing the liquid culture medium (e.g. Soft Agar). Once this has been performed they can be moved either farther away from one another or closer to one another, depending on the spatial distance between the centres of the dishes on the dish support. In one particular variant of this embodiment of the apparatus the spacing means comprise a gear with a fixed gear transmission ratio, which may be such that the ratio of the distances between adjacent pipettes always remains constant. This is an embodiment which is simple from a technical point of view.
According to a further embodiment of the apparatus according to the invention the pipettes are disposable pipettes. This is advantageous in that the respective pipettes can be used only once so that no cross-contamination or mixing of substances may occur.
Further advantageous aspects of the invention become evident from the following description of an embodiment of the invention in which:
In
In a first step S1, the different substance or compound dilutions are prepared and introduced into the wells 10 of the micro-well plate 1. This can be done manually and remote from the apparatus according to the invention, but can also be done in the interior of the outer housing of the apparatus.
The next step S2 will be performed only if it is not the substance or compound per se but rather any potential metabolites thereof which is to be tested. This step S2 comprises the dispensing of a predetermined amount of a liver enzymes mixture into the respective wells 10 of the micro-well plate 1. If the substance per se is to be tested, it is also possible to dispense a predetermined amount of a buffer solution into the respective wells 10 of micro-plate 1.
The next step S3 is capturing and analyzing a picture of the substances or compounds (or their mixtures with liver enzymes or buffer solution, respectively) contained in the wells 10 of the micro-well plate 1. In case a substance or compound precipitates it cannot be absorbed by the bacteria strains, this can only happen when the substance or compound is in solution. Therefore, in case precipitation occurs, the substances contained therein cannot be tested.
The next step S4 comprises dispensing a predetermined amount of a bacteria solution, such as e.g. a solution containing salmonella strains, into the respective wells 10 of the micro-well plate 1.
In a step S5, the so prepared micro-well plate 1 containing in its wells 10 the substance/salmonella mixtures (or the respective substance/salmonella/liver enzymes mixture) is then incubated for a predetermined amount of time under predetermined conditions (and is optionally shaken during that time). Thereafter, the substance/salmonella mixtures (or the respective substance/salmonella/liver enzymes mixtures) are ready to be subjected to the automatic Ames-test with the aid of the apparatus according to the invention, an embodiment of which will be explained further below.
In the next step S6, a number of pipettes 40—in the embodiment described five pipettes—of which only one pipette 40 is shown in
In the next step S7, the pipettes 40 with the intaken substance/salmonella mixtures are moved to a corresponding number of containers where a desired amount of a liquid culture medium 50, such as e.g. a warm Soft Agar solution, is intaken into the pipettes 40 by aspiration. Also, some air may be aspirated, too, so as to achieve a good mixing of the substance/salmonella mixture with the liquid culture medium. The pipettes 40 with the substance/salmonella/culture medium mixture is then moved to a corresponding number of dishes 60, in this embodiment five dishes of which only one dish 60 is shown in
The dishes 60 contain a substantially solid culture medium, such as an Agar disk 61, arranged on the bottom of the respective dish. The dishes 60 in the embodiment described are standard Petri-dishes. Shortly before the dispensing of the mixtures contained in the pipettes 40 into the respective dishes 60, the Agar disks 61 are heated in step S8 with the aid of an IR-radiator so as to increase the hydrophilicity of the Agar disks.
Thereafter, in step S9 the mixture contained in the pipettes 40 is centrally dispensed into the respective dishes 60. The Petri-dishes are then spun either clock-wise or counterclock-wise as is indicated by the arrows so as to coat the solid Agar disk 61 with the dispensed mixture. Spinning is then stopped allowing a uniform film to form on the solid Agar disk 61.
In step S10 a lid is placed onto the Petri-dishes and the Petri-dishes are then incubated for a predetermined amount of time and under predetermined conditions. After that, in step S11 the number of salmonella colonies that have developed is counted in each Petri-dish 60. If no colonies have developed or if only a number of colonies has developed which is lower than a predetermined threshold number, the substance or its metabolite, respectively, has passed the Ames-test.
In
Next, as shown in
The transport means may comprise two laterally arranged belts for moving the Petri-dishes 60 along dish support 6 (see
The lifting members 64 lift the Petri-dishes 60 to the level of the transfer means 65 which move out at the left and right hand sides of the respective lifting members 64. The lifting members 64 are then lowered again so that the Petri-dishes 60 come to rest on the bearing surfaces 650 of the transfer means 65 which are then moved back again to their original position (
The Petri-dishes 60 are introduced into rack 8 from below. Once rack 8 is completely filled with Petri-dishes 60 it can be forwarded to an incubator (not shown) for a predetermined amount of time (e.g. for two days) and under predetermined conditions (e.g. at a temperature of 37° C.) After that, the number of colonies of salmonella strains that have developed during the incubation time is counted, and based on that it is determined whether or not the substance and its metabolite have passed the Ames-test. Therefore, it is vital that for each substance to be tested the substance per se and potential metabolites thereof are subjected to the Ames-test. Once the dispensing of the substances contained in the pipettes 40 has been performed, pipetting unit 4 is moved back towards collecting pan 52. The pipettes 40 used are preferably disposable pipettes 40, so that after single use the used pipettes are inserted into collecting pan 52 and new ones are attached to the pipetting unit 4. The disposable pipettes 40 are preferably made from a suitable (inert) plastic material.
Pipetting unit 4 is shown in more detail in
Also, as is clear from the description of the apparatus 2 while the pipettes 40 have to be arranged at a very small distance relative to one another as they are introduced into adjacently arranged wells 10 of the micro-well plate 1, they have to be moved away from one another so that they have a greater distance relative to one another when they have to be introduced into the containers 5, and they have to be moved still farther away from one another so as to be properly positioned above the respective open Petri-dishes 60. This movement of the pipettes 40 can be achieved with the aid of a spacing mechanism 41, an embodiment of which is shown in
Having described a specific embodiment of the apparatus and process according to the invention it is clear for the skilled person that various modifications can be made without departing from the general teaching of the invention. Therefore, the scope of protection is not intended to be limited to the described embodiments but rather is defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09155692.8 | Mar 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/53605 | 3/19/2010 | WO | 00 | 9/6/2011 |
