Recommendations for Handling the DTP Diversity Set:

**Solubility Issues**

# Background

The Developmental Therapeutics Program of the National Cancer Institute,
through its Drug Synthesis and Chemistry Branch, selected 1,990
compounds as representative of the chemical diversity of the \>140,000
chemicals available for testing in the DTP Pure Compounds Repository.
This group of compounds, termed the "Diversity Set", is maintained in 96
well microtiter plate format, and has been widely distributed to
laboratories engaged in biomedical investigations both within the NIH
and the extramural community, as a research tool for various biological
testing purposes. The compounds, solubilized in neat DMSO at either 10
mM or 1 mM, are stored in freezers until shipped, frozen in a box with
dry ice, to a requestor. Freezer storage of compounds in DMSO is a
generally accepted pharmaceutical industry standard practice in high
throughput screening operations. Additional information about the
Diversity Set can be found at
**http://dtp.nci.nih.gov/branches/dscb/diversity_explanation.html**.

The Natural Products Support Group, SAIC-Frederick, Inc., utilizes the
10mM concentration Diversity Set Plates as Masters for the preparation
of plates for testing laboratories at NCI-Frederick. Visual inspection
of Diversity Set plates which had been in freezer storage for

\~\> 1 year revealed that precipitation of compounds had occurred in
approximately 20% of the wells. A similar problem has not been observed
with those same materials when stored at 1mM.

To measure the molarity of drug remaining in DMSO solution, analysis of
the supernatant liquor from above the solids was performed by
HPLC/ELSD/MS, using freshly solubilized drug from the NCI Repository to
construct a standard curve. Concentrations of drug in solution above
precipitate from individual wells, as determined by area under the curve
when measured by ELSD, ranged from \~8 mM to much less than 0.1 mM, at
least a 2 log difference.

This has raised concerns that, if investigators are unaware of the
possibility of precipitation and other factors related to Diversity Set
plate storage and use and have not taken appropriate measures, assays
will be performed on some compounds at molar concentrations well below
the nominal concentration.

Drugs which have precipitated from 10mM DMSO are very difficult to get
back into solution. Various methods for resolubilization were tried,
including sonicating the plate, shaking, and mild heating. While none of
these methods resulted in the complete resolubilization of all
compounds, vigorous shaking for 20-30 minutes combined with sonication
resulted in the formation of a suspension which can be pipetted for most
problem compounds. Removal of DMSO under high vacuum with no heating
(e.g. Speed Vac) is not difficult. Complete resolubilization of
compounds in plates which had been vacuum dried, into fresh, dry DMSO to
give a 10mM solution, required shaking of the plate for \>12 hours at
room temperature in a dry atmosphere. A greater volume of DMSO per well,
giving a less concentrated solution, will facilitate resolubilization.
When 2 μl of glycerol are added to each well prior to vacuum drying,
thus leaving the drug in a 'glycerol mull' in the well, resolubilization
in DMSO to give a 10mM solution is accomplished in \~\<2 hours of
shaking at room temperature. The individual investigator will have to
determine if the presence of glycerol in his/her specific assay has an
effect.

# Recommendations

Recipients of the Diversity Set should bear in mind that, even when all
compounds are in solution in DMSO when received, individual storage and
use protocols will have a significant effect on the physical condition
of the compounds on the plates. The following factors should be
considered:

-   Storage of Diversity Set plates should be at --70^o^ C or lower.

-   If it is anticipated that multiple uses of a plate set will occur
    over time, consider production of multiple replicate plates the
    first time the Diversity Set plates are allowed to come to room
    temperature. This is because DMSO is a very hygroscopic substance.
    Exposure of DMSO solutions to the atmosphere, as would occur each
    time an aliquot is removed, can quickly result in water gain of
    \>10%, which affects both molarity of drug and tendency for drug to
    precipitate during freezer storage. Thus, as an alternative to
    utilizing a single plate multiple times over a several week period,
    multiple aliquots (e.g., multiple plates @ 5μg/well) taken on the
    first occasion that the Diversity Set plate is allowed to thaw are
    more likely to be accurate than preparing one aliquot each time a
    plate is thawed/frozen.

-   Precipitation is NOT a problem when compound is stored dry in the
    plate. Consider storage of compounds dry, in a freezer, as being
    preferable to solution storage, particularly if long term storage
    and repetitive but infrequent usage is anticipated.

-   A change in the concentration of compounds solubilized into dry DMSO
    was NOT detected in less than one week of freezer storage, but a
    decline in concentration IS detectable for some compounds after two
    weeks. Therefore, to assure the accuracy of the molarity of
    compounds being tested, it would be ideal to bring dry compounds
    into DMSO solution less than one week prior to use. Storage of
    compounds in dry DMSO solution in a dry atmosphere at room
    temperature for a short time (\<1 week) is preferable to freezer
    storage for the same time period, for those compounds likely to
    precipitate.

-   The longer compounds are stored in DMSO, regardless of temperature,
    the more likely it is that precipitation will occur.

-   Precipitation is least likely to be induced when frozen plates are
    thawed quickly and used promptly. If multiple uses from a plate are
    unavoidable, then a "quick thaw/remove seal/sip sample/re-seal/quick
    freeze" in a dry atmosphere should be considered.

-   The presence of water in DMSO significantly depresses the freezing
    point of DMSO. If a researcher prepares a 'stock solution of drug'
    by addition of buffer to a DMSO solution, the freezing point of that
    aqueous/DMSO solution could be as low as --70^o^ C. Clearly
    refrigerator or freezer storage at --20^o^ does not result in a
    frozen solution, and the opportunity for degradation and
    precipitation is increased.

Additional information of solubility and storage of compounds for high
throughput screening is in preparation and will be made available at
**http://npsg.ncifcrf.gov** in the future.

List of compounds which have visibly precipitated in the well from a
10mM DMSO solution during freezer storage:

  ---------------------------- ----------------------- -------------------
  PLATEID                      NSC                     WELLID

  13140000                     9928                    B03

  13140000                     40837                   D05

  13140000                     33145                   H05

  13140000                     48949                   A07

  13140000                     54854                   D08

  13140000                     57884                   E08

  13140100                     151063                  A02

  13140100                     49714                   C02

  13140100                     88418                   D02

  13140100                     30079                   E02

  13140100                     27895                   F02

  13140100                     50570                   G02

  13140100                     88416                   H02

  13140100                     138745                  C03

  13140100                     41629                   D03

  13140100                     88482                   E03

  13140100                     173723                  G03

  13140100                     133887                  G05

  13140100                     155080                  E07

  13140100                     334340                  H07

  13140100                     281668                  G08

  13140100                     310099                  C09

  13140100                     78857                   A10

  13140100                     45074                   D10

  13140100                     47652                   E10

  13140100                     1004                    F10

  13140100                     41355                   B11

  13140100                     145425                  G11

  13140200                     669681                  A02

  13140200                     680927                  B02

  13140200                     52003                   C02

  13140200                     51857                   D02

  13140200                     53396                   E02

  13140200                     74702                   F02

  13140200                     79563                   G02

  13140200                     90318                   H02

  13140200                     90615                   A03

  13140200                     95025                   B03

  13140200                     94783                   C03

  13140200                     109268                  D03

  13140200                     111326                  E03

  13140200                     177395                  F03

  13140200                     169942                  G03

  13140200                     268665                  H03

  13140200                     69343                   B04

  13140200                     77037                   G04

  13140200                     89166                   H04

  13140200                     99804                   D06

  13140200                     182395                  E07

  13140200                     172033                  F07

  13140200                     210595                  B08

  13140200                     210599                  D08

  13140200                     182035                  E08

  13140200                     309881                  A10

  13140200                     310551                  B10

  13140200                     115448                  H10

  13140200                     134755                  B11

  13140200                     203328                  C11

  13140200                     181486                  D11

  13140200                     210627                  F11

  13140200                     267235                  G11

  13140200                     306698                  H11

  13140300                     361814                  B02

  13140300                     343227                  E02

  13140300                     1945                    F02

  13140300                     13359                   G02

  13140300                     1694                    H02

  13140300                     641691                  A03

  13140300                     373529                  E04

  13140300                     372295                  F04

  13140300                     611615                  A06

  13140300                     6176                    E08

  13140300                     6246                    H08

  13140300                     8179                    F09

  13140300                     10776                   E10

  13140300                     11656                   A11

  13140300                     13239                   H11

  13140400                     44251                   A02

  13140400                     18807                   B02

  13140400                     20138                   F02

  13140400                     17776                   G02

  13140400                     27959                   H02

  13140400                     23159                   A03

  13140400                     18451                   B03

  13140400                     16865                   C03

  13140400                     14543                   E03

  13140400                     13581                   F03

  13140400                     15234                   G03

  13140400                     22225                   D04

  13140400                     5159                    G04

  13140400                     13728                   B05

  13140400                     48872                   H05

  13140400                     13579                   B07

  13140400                     48873                   F07

  13140400                     14179                   F08

  13140400                     15226                   A09

  13140400                     15784                   B09

  13140500                     29870                   B02

  13140500                     33389                   C02

  13140500                     38528                   C03

  13140500                     23016                   A05

  13140500                     21518                   F05

  13140500                     28427                   H05

  13140500                     24076                   D06

  13140500                     26669                   H06

  13140500                     26145                   B07

  13140500                     26081                   D07

  13140500                     28527                   H07

  13140500                     29424                   B08

  13140500                     36738                   E09

  13140500                     34444                   F11

  13140600                     42352                   A02

  13140600                     43234                   C02

  13140600                     44138                   D02

  13140600                     51132                   E02

  13140600                     52343                   F02

  13140600                     52389                   D03

  13140600                     39225                   C04

  13140600                     39329                   E04

  13140600                     42656                   F06

  13140600                     46669                   C09

  13140600                     48961                   H10

  13140600                     50145                   F11

  13140700                     58612                   A02

  13140700                     73646                   B02

  13140700                     72942                   C02

  13140700                     86380                   D02

  13140700                     75749                   E02

  13140700                     60036                   G02

  13140700                     67323                   H02

  13140700                     84251                   A03

  13140700                     78043                   B03

  13140700                     57850                   C03

  13140700                     61297                   D03

  13140700                     57788                   A04

  13140700                     57975                   C04

  13140700                     56751                   E04

  13140700                     55462                   H04

  13140700                     54672                   A05

  13140700                     54646                   F05

  13140700                     63331                   C06

  13140700                     66184                   D07

  13140700                     72533                   E07

  13140700                     73389                   F07

  13140700                     66185                   E08

  13140700                     77597                   F09

  13140700                     80737                   H09

  13140700                     85359                   G11

  13140800                     96396                   A02

  13140800                     101825                  B02

  13140800                     92601                   C02

  13140800                     87450                   D02

  13140800                     96606                   E02

  13140800                     92988                   F02

  13140800                     93967                   G02

  13140800                     96682                   H02

  13140800                     112751                  E03

  13140800                     88795                   E05

  13140800                     88999                   G05

  13140800                     95865                   E06

  13140800                     91750                   G06

  13140800                     98019                   G08

  13140800                     105550                  C09

  13140800                     101824                  G09

  13140800                     106958                  H09

  13140800                     101548                  B10

  13140800                     101984                  C10

  13140800                     105900                  E10

  13140800                     109174                  F11

  13140900                     151831                  A02

  13140900                     137784                  B02

  13140900                     128884                  C02

  13140900                     133811                  D02

  13140900                     133896                  F02

  13140900                     154652                  G02

  13140900                     164460                  E03

  13140900                     114528                  A04

  13140900                     114743                  B05

  13140900                     114831                  D05

  13140900                     125626                  F05

  13140900                     133593                  H05

  13140900                     133594                  B06

  13140900                     132498                  C06

  13140900                     134667                  G07

  13140900                     140899                  H07

  13140900                     142269                  G08

  13140900                     143019                  H08

  13140900                     134664                  D09

  13140900                     168467                  G09

  13140900                     153330                  A10

  13140900                     150169                  D10

  13140900                     150554                  E10

  13140900                     150080                  F10

  13141000                     294149                  B02

  13141000                     314885                  D02

  13141000                     320514                  F02

  13141000                     328200                  G02

  13141000                     322920                  H02

  13141000                     329070                  D03

  13141000                     321518                  G03

  13141000                     170578                  B04

  13141000                     183519                  D05

  13141000                     190577                  G05

  13141000                     191260                  H05

  13141000                     201980                  F06

  13141000                     210282                  F07

  13141000                     247535                  D08

  13141000                     271928                  A09

  13141000                     288391                  F09

  13141000                     294153                  G09

  13141000                     303530                  E10

  13141000                     303851                  F10

  13141000                     305743                  G10

  13141000                     306960                  H10

  13141100                     372045                  C02

  13141100                     344028                  D02

  13141100                     95609                   E02

  13141100                     360211                  G02

  13141100                     668414                  B03

  13141100                     669581                  D03

  13141100                     274547                  E03

  13141100                     306711                  F03

  13141100                     23128                   G03

  13141100                     93352                   H03

  13141100                     332429                  A04

  13141100                     343548                  F04

  13141100                     351520                  E05

  13141100                     361672                  F06

  13141100                     364164                  G06

  13141100                     379553                  H06

  13141100                     409455                  F07

  13141100                     602670                  D08

  13141100                     403251                  G08

  13141100                     36806                   B10

  13141100                     72938                   D10

  13141100                     270718                  E10

  13141100                     676693                  A11

  13141100                     614846                  C11

  13141100                     624983                  D11

  13141100                     625169                  E11

  13141200                     12363                   A02

  13141200                     12339                   C02

  13141200                     7345                    D02

  13141200                     169534                  E02

  13141200                     12492                   F02

  13141200                     5844                    G02

  13141200                     307241                  H02

  13141200                     13705                   B03

  13141200                     1614                    D03

  13141200                     4292                    E03

  13141200                     10580                   G03

  13141200                     3323                    H04

  13141200                     5069                    G06

  13141200                     4895                    H06

  13141200                     7520                    F07

  13141200                     10424                   H07

  13141200                     12155                   H08

  13141200                     12666                   H09

  13141200                     13161                   C10

  13141200                     13791                   H10

  13141300                     14163                   A02

  13141300                     14410                   B02

  13141300                     14703                   C02

  13141300                     15603                   G02

  13141300                     18695                   D03

  13141300                     18758                   E03

  13141300                     24859                   H03

  13141300                     14143                   D04

  13141300                     16211                   H04

  13141300                     17474                   E05

  13141300                     19480                   C06

  13141300                     23173                   H06

  13141300                     23583                   A07

  13141300                     24048                   G07

  13141300                     28311                   H09

  13141300                     13987                   E10

  13141300                     16087                   G10

  13141300                     17600                   B11

  13141300                     23217                   G11

  13141300                     28136                   H11

  13141400                     45572                   A02

  13141400                     35682                   B02

  13141400                     37245                   D02

  13141400                     35049                   E02

  13141400                     38347                   F02

  13141400                     33571                   G02

  13141400                     40016                   H02

  13141400                     45238                   A03

  13141400                     34238                   B03

  13141400                     37049                   E03

  13141400                     33052                   A04

  13141400                     31303                   H04

  13141400                     36369                   F06

  13141400                     44480                   A08

  13141400                     44585                   D08

  13141400                     47118                   D10

  13141400                     45730                   H10

  13141500                     62594                   B02

  13141500                     54340                   D02

  13141500                     49487                   F02

  13141500                     63875                   G02

  13141500                     48300                   A03

  13141500                     62586                   C03

  13141500                     64337                   D03

  13141500                     48630                   E03

  13141500                     54101                   F03

  13141500                     48223                   G03

  13141500                     57443                   H03

  13141500                     59620                   A04

  13141500                     48858                   D04

  13141500                     48448                   E04

  13141500                     48151                   F04

  13141500                     48874                   G04

  13141500                     56624                   H04

  13141500                     64678                   A05

  13141500                     64814                   B05

  13141500                     56998                   E05

  13141500                     50567                   F05

  13141500                     62349                   H05

  13141500                     57998                   F06

  13141500                     62598                   E07

  13141500                     64408                   G07

  13141500                     48388                   H07

  13141500                     47938                   H08

  13141500                     47949                   A09

  13141500                     48684                   H09

  13141500                     56681                   E10

  13141500                     56939                   F10

  13141500                     52929                   G10

  13141500                     54278                   F11

  13141600                     65933                   A02

  13141600                     79050                   C02

  13141600                     87015                   E02

  13141600                     66207                   G02

  13141600                     72914                   H06

  13141600                     73100                   A07

  13141600                     73254                   C07

  13141600                     75503                   H07

  13141600                     77554                   F08

  13141600                     80177                   F09

  13141600                     81540                   A10

  13141600                     84093                   H10

  13141600                     85459                   G11

  13141600                     85479                   H11

  13141700                     92588                   C02

  13141700                     94820                   F02

  13141700                     95926                   H02

  13141700                     106221                  D03

  13141700                     109451                  G03

  13141700                     108098                  H03

  13141700                     95916                   F05

  13141700                     97999                   C07

  13141700                     98905                   E07

  13141700                     99504                   F07

  13141700                     99515                   G07

  13141700                     99526                   H07

  13141700                     99663                   F08

  13141700                     99671                   E09

  13141700                     109131                  H10

  13141700                     109188                  A11

  13141700                     109248                  C11

  13141700                     92585                   E11

  13141700                     99445                   F11

  13141800                     110380                  A02

  13141800                     113491                  B02

  13141800                     117271                  D02

  13141800                     114792                  E02

  13141800                     121865                  G02

  13141800                     118516                  H02

  13141800                     122140                  A03

  13141800                     121304                  B03

  13141800                     120686                  C03

  13141800                     118207                  D03

  13141800                     116654                  E03

  13141800                     116709                  G03

  13141800                     119236                  H03

  13141800                     121860                  A04

  13141800                     121838                  B04

  13141800                     117489                  C04

  13141800                     116702                  D04

  13141800                     109816                  F04

  13141800                     114382                  G04

  13141800                     109509                  H04

  13141800                     118836                  C05

  13141800                     113909                  H06

  13141800                     109593                  D07

  13141800                     116491                  F07

  13141800                     120672                  H08

  13141800                     120688                  F09

  13141800                     118176                  D11

  13141900                     125252                  A02

  13141900                     131612                  C02

  13141900                     135882                  D02

  13141900                     136469                  E02

  13141900                     157469                  F02

  13141900                     163865                  G02

  13141900                     163443                  B03

  13141900                     168468                  E03

  13141900                     122370                  A04

  13141900                     123111                  F04

  13141900                     125095                  F05

  13141900                     125908                  G05

  13141900                     126347                  H05

  13141900                     126401                  A06

  13141900                     126710                  B06

  13141900                     130796                  B07

  13141900                     131614                  F07

  13141900                     132230                  B08

  13141900                     133488                  F08

  13141900                     164137                  H08

  13141900                     134148                  A09

  13141900                     136472                  F09

  13141900                     136473                  G09

  13141900                     162215                  E10

  13141900                     164640                  D11

  13142000                     209910                  A02

  13142000                     209931                  B02

  13142000                     169930                  C02

  13142000                     299137                  D02

  13142000                     169931                  F02

  13142000                     169925                  G02

  13142000                     169936                  H02

  13142000                     309895                  A03

  13142000                     297813                  B03

  13142000                     170561                  H03

  13142000                     216633                  A04

  13142000                     292213                  B04

  13142000                     295642                  E04

  13142000                     294526                  H05

  13142000                     250826                  E06

  13142000                     270156                  F06

  13142000                     270737                  G06

  13142000                     311152                  E08

  13142000                     310366                  F08

  13142000                     305831                  C09

  13142000                     309883                  H09

  13142000                     170992                  D10

  13142000                     268879                  A11

  13142000                     216621                  B11

  13142000                     255980                  H11

  13142100                     319464                  A02

  13142100                     327702                  C02

  13142100                     327704                  D02

  13142100                     371884                  F02

  13142100                     372037                  G02

  13142100                     372046                  H02

  13142100                     372060                  A03

  13142100                     372618                  B03

  13142100                     379640                  C03

  13142100                     625324                  E03

  13142100                     381469                  F03

  13142100                     332471                  H03

  13142100                     319440                  G04

  13142100                     319447                  H04

  13142100                     321496                  A06

  13142100                     321578                  C06

  13142100                     326182                  F06

  13142100                     328087                  A07

  13142100                     339585                  F07

  13142100                     372074                  G07

  13142100                     372280                  A08

  13142100                     372768                  C08

  13142100                     373054                  F08

  13142100                     373058                  G08

  13142100                     373989                  H08

  13142100                     607796                  A11

  13142100                     629659                  F11

  13142100                     327705                  H11

  13142200                     680517                  A02

  13142200                     521717                  D02

  13142200                     81670                   E02

  13142200                     159165                  B03

  13142200                     241261                  E03

  13142200                     659162                  F03

  13142200                     639174                  E04

  13142200                     643735                  H04

  13142200                     657704                  F05

  13142200                     659694                  A06

  13142200                     629974                  G10

  13142200                     634396                  H10

  13142200                     634663                  A11

  13142200                     658142                  E11

  13142300                     350363                  C02

  13142300                     360494                  D02

  13142300                     361815                  E02

  13142300                     152432                  G02

  13142300                     618077                  H02

  13142300                     41458                   A03

  13142300                     83628                   B03

  13142300                     117212                  C03

  13142300                     357650                  G03

  13142300                     351123                  H03

  13142300                     138756                  A04

  13142300                     141562                  B04

  13142300                     363916                  C04

  13142300                     339908                  H04

  13142300                     136476                  D05

  13142300                     363935                  E05

  13142300                     369205                  G05

  13142300                     371684                  H05

  13142300                     141566                  C06

  13142300                     147744                  F06

  13142300                     148354                  H06

  13142300                     150289                  A07

  13142300                     157307                  H08

  13142300                     351351                  C11

  13142400                     122405                  A02

  13142400                     118217                  B02

  13142400                     187613                  D02

  13142400                     115883                  E02

  13142400                     175423                  F02

  13142400                     176327                  G02

  13142400                     201430                  H02

  13142400                     179187                  A03

  13142400                     194598                  C03

  13142400                     201873                  E03

  13142400                     116064                  F03

  13142400                     117198                  C04

  13142400                     120639                  H05

  13142400                     172614                  H06

  13142400                     175650                  C07

  13142400                     176328                  C08

  13142400                     177407                  H08

  13142400                     201868                  B09

  13142400                     188491                  C10
  ---------------------------- ----------------------- -------------------