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An t i- Can c e r I

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Margaretenstr. 7/2 Tel.: +43 (1) 5861223

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1040 Vienna, Austria Fax: +43 (1) 5868994

UKRAINIAN ANTI-CANCER INSTITUTE


nowicky@ukrin.com, http://www.ukrin.com

NSC 631570 (UKRAIN)


EFFICACY,
SAFETY,
QUALITY

Vienna, 7.3.2011
NSC631570(UKRAIN):EFFICACY,SAFETY,QUALITY

INTRODUCTION:THEFIRSTMEDICAMENTTHATKILLSCANCERCELLSBUTNOTHEALTHYCELLS
___________________________________________________________________________ 3
Generaloncancertreatment ___________________________________________________ 3
Cancercellscanbekilledwithoutdamaginghealthycells ____________________________ 4
AffinityofNSC631570tothecancercells _________________________________________ 8
Immunemodulatingproperties _________________________________________________ 9
Theangiogenicproperties ____________________________________________________ 12
I.EFFICACY _________________________________________________________________ 13
PhaseIClinicalStudy_________________________________________________________ 13
TheDoseFindingStudy(PhaseII)_______________________________________________ 13
PhaseIIIClinicalTrials ________________________________________________________ 14
Pancreaticcarcinoma ________________________________________________________ 15
Colorectalcancer____________________________________________________________ 16
Prostatecancer _____________________________________________________________ 17
Breastcancer _______________________________________________________________ 17
Bladdercancer______________________________________________________________ 18
Malignantmelanoma ________________________________________________________ 18
Braintumors _______________________________________________________________ 18
Malignantgynaecologictumors ________________________________________________ 19
TheselectiveaccumulationofNSC631570inthetumortissueprovendueto
autofluorescence____________________________________________________________ 21
CASEREPORTS ______________________________________________________________ 21
Breastcancer ____________________________________________________________________ 21
Sarcomas _______________________________________________________________________ 21
Renalcellcarcinoma ______________________________________________________________ 24
Testicularcancer _________________________________________________________________ 24
Esophaguscarcinoma _____________________________________________________________ 24
Bladdercancer(urothelialcellcarcinoma)_____________________________________________ 24
Neuroblastoma __________________________________________________________________ 24
Hereditarydiseases_______________________________________________________________ 24
ANTIVIRALPROPERTIES ______________________________________________________ 27
THEINHIBITIONOFTHETUMORALANGIOGENESIS ________________________________ 27
THESELECTIVEEFFECTOFNSC631570___________________________________________ 32
InductionoftheApoptosisinCancerCells_____________________________________________ 34
InhibitionoftheTubulinPolymerisation ______________________________________________ 35
ActivationofMitochondrialCaspases ________________________________________________ 36
TheEffectonCyclinsandCyclinͲDependantKinases_____________________________________ 36
TheEffectontheExpressionofhENT1anddCK ________________________________________ 37
TheEffectontheDNAandProteinSynthesis __________________________________________ 37
TheEffectontheProteinsInvolvedintotheRemodellingofExtracellularMatrix _____________ 37

1
TheEffectontheHeatShockProteins ________________________________________________ 39
TheEffectontheElectrokineticPotential _____________________________________________ 39
THEINVIVOSTUDIES ________________________________________________________ 39
TheEffectontheGrowthofMelanomaB16inMice_____________________________________ 40
TheEffectonCysteinProteases _____________________________________________________ 40
MODULATIONOFTHEIMMUNESYSTEM __________________________________________ 41
ClinicalImmunology __________________________________________________________ 42
TheEffectontheDendriticCells _________________________________________________ 42
ImmunologicalpropertiesofUkrain(NSC631570) ___________________________________ 43
NSC631570modulatesthenaturaldefensesystemagainstdevelopingcancer _____________ 43
TheStudiesinvitroandinvivo __________________________________________________ 44
RADIOPROTECTIVEEFFECT ____________________________________________________ 45
TOXICOLOGICSTUDIES ________________________________________________________ 46
NORMALISATIONOFTHEMETABOLISM _________________________________________ 47
BoneMetabolismandOsteoporosis ___________________________________________________ 47
THEEFFECTONVARIOUSENZYMES ______________________________________________ 48
INTERACTIONWITHOTHERDRUGS _____________________________________________ 49
NonͲOpioidAnalgesics ____________________________________________________________ 49
OpioidAnalgesics _________________________________________________________________ 49
Streptozotocin ___________________________________________________________________ 50
PorphyrinDerivates ______________________________________________________________ 50
Anticonvulsants__________________________________________________________________ 50
INTERACTIONWITHOTHERTHERAPYMODALITIES ________________________________ 50
Localhyperthermia _______________________________________________________________ 50
EndovascularLaserTherapy/PhotodynamicTherapy______________________________________ 51
Ozone __________________________________________________________________________ 51
Others _________________________________________________________________________ 51

II.SAFETY_______________________________________________________________ 52
AcuteIntravenousToxicitywithRats _________________________________________________ 52
AcuteIntravenousToxicitywithMice ________________________________________________ 52
AcuteIntramuscularToxicitywithRats _______________________________________________ 53
AcuteOralToxicityStudywithRats __________________________________________________ 53
LocalTolerabilityStudy____________________________________________________________ 53
MicronucleusTestwithMice _______________________________________________________ 54
SalmonellatyphimuriumReverseMutationTest _______________________________________ 54
LiverToxicity ____________________________________________________________________ 54
OtherToxicityStudies _____________________________________________________________ 55
PHARMACOKINETICS_________________________________________________________ 56
BIOPHYSICALPROPERTIES ____________________________________________________ 56
III.QUALITY ____________________________________________________________ 57
GreatercelandineͲExtractfromtheEuropeanPharmacopoeia6.0 ___________________ 57
Thiotepa–ExtractfromtheUSPharmacopoeia,24thedition ________________________ 59
Stability ___________________________________________________________________ 60
BIBLIOGRAPHY __________________________________________________________ 61

2
INTRODUCTION:THEFIRSTMEDICAMENTTHATKILLSCANCERCELLSBUTNOTHEALTHY
CELLS


Generaloncancertreatment

Surgery, chemotherapy and radiotherapy are the three most important types of cancer
treatment. However, each of them have their limitations and as well as considerable
adverseeffects.Surgeryistheoldesttreatmentoptionand,ifpossible,isstillfrequently
themethodoffirstchoice.Unfortunatelycurativesurgicalinterventionisonlypossiblein
few cases; the residual tumour usually remains unrecognised, leading to tumour
recurrenceandmetastazing.Additionaltreatmentthencomesintoconsideration.
Bothradiationandchemotherapydonothaveaselectiveeffectagainsttumourcells,they
are themselves carcinogenic (they cause cancer) and mutagenic (they damage
chromosomesandchangethegeneticmakeͲup).
Radiation therapy works by damaging the DNA of cancerous cells. This DNA damage is
then passed on through cell division, accumulating damage to the cancer cell's DNA,
causingthemtodieorreproducemoreslowly.Radiotherapycausesvaryingsideeffects
duringtreatment(acutesideeffects),inthemonthsoryearsfollowingtreatment(longͲ
termsideeffects),orafterreͲtreatment(cumulativesideeffects).
Thefirstuseofdrugstotreatcancerwasintheearly20thcentury.Mustardgaswasused
as a chemical warfare agent during World War I and was studied further during World
War II. During a military operation, a group of people were accidentally exposed to
mustard gas and were later found to have very low white blood cell counts. It was
reasonedthatthisagentmighthaveasimilareffectoncancer.In1942mechlorethamine
was first used as a cytostatic agent for cancer therapy. Most commonly, chemotherapy
acts by killing cells that divide rapidly, one of the main properties of most cancer cells.
Thismeansthatitalsoharmscellsthatdividerapidlyundernormalcircumstances:cellsin
thebonemarrow,digestivetractandhairfollicles;thisresultsinthemostcommonside
effectsofchemotherapy:myelosuppression(decreasedproductionofbloodcells,hence
also immunosuppression), mucositis (inflammation of the lining of the digestive tract),
and alopecia (hair loss). Moreover, various groups of cytostatic agents cause other
specificadverseeffects.
Complex concomitant therapies have being used to alleviate adverse effects of the
chemotherapy, i.e. corticosteroids, antiemetic agents etc. Still, a part of chemotherapy
cyclesmustbeperformedwithreduceddose,postponedorstopped.
Itisclearthattheproblemoftheimmenseadverseeffectsofchemotherapycannotbe
solved in a customary way. This could only be achieved by preparations which only kill
cancercellswhileleavinghealthycellsundamaged,inotherwordswhichactselectively
againstcancercells.
With the new generation of ‚targeted’ anticancer agents one tries to block specific
molecules needed for tumor development and growth. Still, current results of
chemotherapyareunsatisfactory.Cancerisresponsiblefor20,000deathsperdayacross
the world, or 7.6 million people a year, according to a report of the American Cancer
Society(http://www.cancer.org/Research/CancerFactsFigures/index).

3
It is evident that it has always been the greatest wish of all scientists and cancer
researchers to find a preparation which kills only cancer cells and leaves healthy cells
undamaged.Inotherwords,apreparationwithaselectiveeffectonlyagainstcancercells
but not against healthy cells. All the efforts of scientists all over the world have been
unsuccessful–therewasthestrongconvictionthatthisproblemwasinsolublebecause
thedifferencebetweenahealthycellandacancercellistoosmall.


Cancercellscanbekilledwithoutdamaginghealthycells

Anewpreparationwaspresentedatthe13thInternationalCongressofChemotherapyin
Vienna in AugustͲSeptember 1983 – thiophosphoric acid derivative of alkaloids from
greatercelandine(NSC631570,tradenameUkrain).Thedevelopmentofthispreparation
wasthefirstsignificantsteponthewaytosolvingtheproblem.Invitrotestsshowedthat
after incubation with NSC631570 normal liver cells and Ehrilch tumour ascitic cells
demonstratedifferentoxygenconsumption:afterinitialincrease,oxygenconsumptionin
cancercellsfallstozerowhereasoxygenconsumptioninnormalcellsreturnstonormal
and the cells remain undamaged (38). This study brought the first indications that, in
contrast to its starting substances thiotepa (a well known cytostatic agent) and greater
celandine alkaloids, NSC631570 is in fact only toxic against cancer cells and not against
normalcells.
The second indication was provided by clinical use, where NSC631570 caused no
noteworthy sideͲeffects (21). It improved patients’ general condition as well as their
immunestatuswhichhadpreviouslybeenimpairedbychemotherapy(22).
The third indication was provided by a study at the University of Miami, based upon
which the therapeutic index of NSC631570 was calculated to be 1250 (26). This is
unusually high for an antiͲcancer preparation. The therapeutic index is the ratio of the
toxic dose to the therapeutic dose and reflects the level of safety of a medicine. The
therapeutic index of conventional cytostatic preparations is in the range of 1. 4Ͳ1.8
meaningthatanoverdosecanhavefatalconsequences.Thereisnoriskofanoverdosage
withNSC631570onaccountofitsveryhightherapeuticindexof1250.
ThedevelopmentofNSC631570wasatrailͲblazingdiscovery.Thepreparationhasshown
thattheproblemcanbesolvedandhaschangedourideasabouthealthyandcancercells.
The presentation at the congress was greeted both by scepticism and great interest.
ManyreputedresearchinstitutessuchastheNationalCancerInstitute(USA),EORTC,the
University of Miami, the Rochester University (USA), and University of Tübingen
(Germany) began to test the preparation to better explain its unique properties and
anticancer potential. In the NCI test model, in contrast to conventional cytostatic
preparations which caused the growth inhibition only in some cancer cell lines, like
thiotepa inhibited the growth of MLIͲ09 (nonͲsmall lung cancer) and UOKͲ57LN (renal
cancer),NSC631570killedall60testedcancercelllines(190),whichrepresenttheeight
importanthumantumours,includingthecelllineswhichwereresistanttothestrongest
cytostaticdrugatthetime,cisplatin.
This generated still more interest in scientific circles. Leading scientists examined
NSC631570, each group with the method available. Thanks to this variety of

4
experimentationthefinemechanismsofactionofNSC631570atdifferentlevelscouldbe
deciphered:firstlyatcellularlevelwithoxygenconsumption(effectonmitochondria;38),
then on the level of chromosomes (effect on DNS and RNS; 58, 63), cell organelles and
molecules (43, 62). These experiments produced extremely interesting results and not
onlyconfirmedtheselectiveeffectofNSC631570butthoroughlydestroyedalldoubton
thematter.ThismeansthatNSC631570candifferentiatebetweenhealthyandmalignant
cells, a feature not yet managed by other antiͲcancer drugs. Interest in NSC631570 is
growingandresearchiscontinuing(261Ͳ266).
Researchers at the University of Natural Resources and Life Sciences, Vienna compared
NSC631570’sinhibitiveeffectonthereproductionofmalignantandnormalcells.Inorder
to achieve 50% growth inhibition a tenfold concentration of NSC 631570 had to be
administered to normal endothelial cells in comparison to a human osteosarcoma cell
line. Laser scanning microscopy showed a high capacity to absorb NSC631570 in
malignantcellswhileabsorptioninnormalcellswasconsiderablelowerunderthesame
experimentalconditions(36).
In a study of the effectof NSC631570 on KͲ562 erythroleucemiacells it was found that
thepreparationcausesbimodalcelldeath.AtlowerNSC631570concentrationsmalignant
cellsdieasaresultofapoptosis,athigherconcentrationstheformationofmicrotubules
ispreventedundpolyploidyoccurs(62).
In 1998 a group around Anne Panzer (University of Pretoria, South Africa) proved the
selectiveeffectofNSC631570onmolecularlevel.Testsonhumancervicalcarcinomacells
HeLa,squamouscellcarcinomaWHCO5andnormalequinelungcelllinesdemonstrated
thatNSC631570isselectivelytoxicagainstcancercells.Itcausesametaphaseblockwhich
is characterised by an abnormal distribution of chromosomes and the formation of
micronucleiandresultsinapoptosis.Normalcellsarenotinfluencedintheprocess(139).
In 2000 in a study of cell proliferation after absorption of BrdU in the cell lines AsPC1,
BxPC3,MiaPaCa2,JurkatandTHPͲ1andthecellcyclephases–withthehelpofGiemsa
staining, researchers from Ulm found that 10 µg/ml NSC631570 causes a clear
accumulationofcancercellsinphaseG2/M.Interestinglynodifferencewasseeninthe
rate of apoptosis in normal peripheral mononuclear cells treated with NSC631570
compared to those untreated. The blastogenic response of mitogenͲstimulated
lymphocytes was even significantly increased. The authors showed that NSC631570
blocks pancreas cancer cells in the prophase by inhibiting tubulin polymerisation (181).
ThisstudyconfirmedthatNSC631570exertsnoinfluenceonnormalcells.
Also in 2000 researchers at Rochester University (USA) examined the effect of NSC
631570 on concentrations of cyclins and cyclinͲdependent kinases in epidermoid
carcinoma cell lines ME180 and A431 as well as the prostate cancer cell line LNCaP.
ChangeswerefoundintheconcentrationsofmitoticcyclinsAandB1aswellasCDK1and
CDK2. The researchers also observed increased expression of the CDKͲinhibitor p27 in
bothcelllines,whichledtotheaccumulationofcancercellsbutnotnormalcellsinthe
G2/Mphase(147,149).
In another study from 2000 entitled „NSC631570TM, a semisynthetic Chelidonium majus
alkaloidderivative,actsbyinhibitionoftubulinpolymerizationinnormalandmalignantcell
lines“ (Cancer Letters 160 (2000) 149Ͳ157)  researchers from South Africa found that

5
NSC631570 inhibits the polymerisation of tubulin (185). The authors used human
fibroblastsasanormalcellline.
NormalhumanfibroblastswerelaterusedinthetestsbythescientistsoftheEberhardͲ
KarlsͲUniversityTuebingen(Germany)aswellasoftheInstitutoNacionaldeCancerologia
(Mexiko City, Mexiko), also. Both research teams could not find any toxic effects of
NSC631570onthesenormalcelllines.Moreover,A.Panzeretalnotedintheirarticle:„Both
NSC631570TMandchelidoninehadweakactivityinthissystem…“(185).


100

90
80
% cells in G2/M phase

70
60 NeoK
50 A431
40 ME180

30

20
10

0
0 3,5 7 17 35
Drug concentration, µM

Fig.1.EffectofdifferentNSC631570concentrationsonG2/Mcellpopulation.Cellsweretreated
withtheindicatedconcentrationsfor24handanalyzedforDNAcontentbyflowcytometry.The
data presented are the percentage of NeoK (normal human keratinocytes), A431 and ME180
(epidermoidcarcinomacells)cellpopulationsinG2/Mphase.FromRoublevskaiaetal,2000(147).

In 2002 scientists at the EberhardͲKarlsͲUniversity Tuebingen (Germany) examined the
effect of NSC 631570, alone or combined with radiation (1Ͳ10 Gy), on cell survival, the
modificationofthecellcycleandtheinductionofapoptosisintheexponentiallygrowing
human tumour cell lines MDAͲMBͲ231 (breast cancer), PAͲTUͲ8902 (pancreatic cancer),
CCLͲ221(coloniccancer),UͲ138MG(glioblastoma)andthehumanskinfibroblastsHSF1,
HSF2andlungfibroblastsCCD32ͲLU.WithoutradiationNSC631570hadatimeanddose
dependent cytotoxic effect which was more pronounced in cancer cells than in normal
cells. Combined with radiation NSC 631570 resulted in increased cytotoxicity against
colonic cancer and glioblastoma cell lines but not against breast cancer and pancreatic
cancercelllines.BymeansofflowcytometryitwasshownthatNSC631570modulated
the toxic effect of radiation on these human cancer cell lines by causing their
accumulationintheG2/Mphaseofthecellcycle.Itsprotectiveeffectonnormalhuman
fibroblastsspeaksinfavourofitsuseincombinedradioͲchemotherapy(184).
In 2005 the ability of NSC631570 to induce apoptosis was studied at the University
HospitalTuebingen(Germany)onaJurkatlymphomamodel.NSC631570proveditselfto

6
beastronginducerofapoptosis.Moredetailedinvestigationsshowedthatitcausedthe
depolarisation of mitochondrial membranes and as a result the activation of caspases
(246).


0,9

0,8
0,7
Cell survival

0,6
R
0,5
R+U
0,4
0,3

0,2

0,1

0
CCL-221 U-138MG HSF1 HSF2
Cell lines

Fig.2.SurvivalofirradiatedcellswithandwithoutNSC631570treatment.R–irradiation,2Gy;R+
U–irradiation,2GyandtreatmentwithNSC631570.Celllines:CCLͲ221Ͳcolorectalcarcinoma,UͲ
138MGͲglioblastoma,HSF1undHSF2Ͳnormalfibroblasts.AfterCordesetal,2002(184).

In2006researchersattheInstitutoNacionaldeCancerologia(MexicoCity,Mexico)found
that NSC631570 triggers apoptosis in a series of cancer cell lines (human cervical
carcinomaHeLa,HeKB,HeKS32,HeBcll3,HeNFRandHelKK,coloniccancerSW480,kidney
cancer HEK293, osteosarcoma MG 63) by activating the intrinsic cell death pathway.
InterestinglythenonͲtransformedfibroblastcelllinehTERTwasnotsensitivetothisdrug
(255).
AttheUniversityofPisa(Italy)thecytotoxiceffectofNSC631570wasexaminedontwo
primary pancreatic cancer cell lines (PPTCC), fibroblasts from ductal pancreatic cancer
tissuesamples(FͲPDAC)andanimmortalisedductalepithelialpancreascellline(HPNE).
CytotoxicitywasestablishedbymeansofCellTiter96kit.ThemodulationofNSC631570
absorption in the medium was determined with the help of the fluorescence of NSC
631570underUVlight.ThecytotoxiceffectofNSC631570onpancreaticcancercelllines
wasconsiderablyhigherthanonthefibroblastsandepithelialcells(20%asagainst80%
livingcells).InadditionthefluorescencetestshowedthatPPTCCcellsabsorbedmoreof
thepreparationthanFͲPDACandHPNEcells.TheseresultsshowaselectiveeffectofNSC
631570 on the pancreatic cancer cells, which indicates different transport systems or a
higherrateofmetabolismofthepreparationinthePDACcells(265).
Ascanbeseen,manyaspectsoftheeffectofNSC631570oncancerandnormalcellshave
sofarbeenstudied.Nevertheless,thepossibilityexiststhattheseeffectsaremerelythe
resultsofanasyetunknownprocesswhichNSC631570inducesincancercellsbutnotin
normal cells. As NSC631570 killed in the tests almost all cancer cell lines, it can be
suggested that a factor occurs when a normal cell becomes a cancer cell. This factor is

7
affected with NSC631570. If this phenomenon can be deciphered it could provide very
importantindicatorsforthesignificantdifferencebetweennormalcellsandcancercells,
pointtowardstherealcauseofcancerandopenupcompletelynewperspectivesforthe
developmentofnewantiͲcancerdrugs,notonlyfortreatmentbutalsoforuseincancer
prevention.


100
90
80

70
% Viability

60
HeLa
50
hTERT
40
30

20
10

0
50 25 10 5
Cellular dencity, cells x 1000/cm2

Fig. 3. Effects of NSC631570 on cervical carcinoma cell line HeLa and on the normal fibroblasts
hTERT expressed as percentage of living cells after 48 h incubation with NSC631570 40 µg/ml.
FromMendozaetal,2006(255).

These studies on the selective effect of NSC631570 clearly revealed cancer cells can be
killedwithoutanydamagetothehealthtissues.


AffinityofNSC631570tothecancercells

It is well known all living cells have a membrane potential of about Ͳ60 to –100mV. The
negative sign of the membrane potential indicates that the inside surface of the cell
membrane is relatively more negative than the than the immediate exterior surface of
thecellmembrane.
As far back as 1938 Dr. Paul Gerhardt Seeger originated the idea that destruction or
inactivation of enzymes, like cytochrome oxidase, in the respiratory chain of the
mitochondriawasinvolvedinthedevelopmentofcancer.
1948G.R.MiderandcoͲworkersrevealedthecancercellsaremorenegativelychargedthan
normalcells.
During the production of NSC631570 celandine alkaloids become positively charged. This
explainsthehighaffinityofNSC631570tothecancercells.
Researchers at the University of Natural Resources and Life Sciences, Vienna (Austria)
revealedhumanosteosarcomacellstohaveahighcapacitytoabsorbNSC631570while

8
absorption in normal endothelial cells was considerable lower under the same
experimentalconditions(36).
ThisselectiveuptakeofNSC631570inthecancercellshasbeenconfirmedduetoitsunique
propertytoautofluorescenceunderUVlight(5).
AstudyexaminedhowNSC631570affectstheelectrokineticpotential(EKP)ofmalignant
andbenigncells.TheEKPoftheEhrlich’scarcinomacellsdroppedafterincubationwith
NSC631570.TheEKPdecreaseofnormalcellswaslesspronounced(221).
ThecytotoxiceffectofNSC631570wasexaminedontwoprimarypancreaticcancercell
lines (PPTCC), fibroblasts from ductal pancreatic cancer tissue samples (FͲPDAC) and an
immortalisedductalepithelialpancreascellline(HPNE).Cytotoxicitywasestablishedby
meansofCellTiter96kit.
In the tests with primary pancreatic cancer cell lines the modulation of NSC 631570
absorption in the medium was determined with the help of the fluorescence of NSC
631570underUVlight.Thefluorescencetestshowedthatcarcinomacellsabsorbedmore
ofthepreparationthanfibroblastsandnormalepithelialcells(265).
The selective accumulation of NSC 631570 in the cancer cells explains its favourable safety
profileandhightherapeuticindexof1250.


Immunemodulatingproperties

Unusual for an anticancer agent NSC 631570 possesses some distinct immune properties
(24,44).ItwasProf.AndrejsLiepinsoftheSt.John’sMemorialUniversity,St.John’s,Canada
whofirstpointedtothisinterestingfact.IntheworkwiththeC57BL/6miceherevealed
NSC631570tobeaneffectivebiologicalresponsemodifier(BRM).Afterincubationwith
NSC631570 the lytic activity of the splenic lymphocytes from the alloimmunised mice
increased up to 48 fold (fig. 4). On the day 18 this increase was the highest and then
fadedslightly(fig.5).ThelyticactivityoftheinterleukinͲ2treatedspleencellsandofthe
peritonealexudatelymphocytesincreasedaswell(17).
InseveralimmunetargetͲeffectorsystemsNSC631570significantlyamplifiedthemalignotoxic
activity of macrophages (231), lymphocytes and NK cells (47), and stimulates dendritic cells
maturationinvitro(258).WhiletheparameterslikeBͲlymphocytescount,immuneglobulin
concentrations,complementandacutephaseproteinsdidnotchangedsignificantly,itcanbe
postulated NSC 631570 modulates the cellular part of the immune system whereas the
humoralpartremainsunaffected.
Humanlymphocytesaswellasguineapiglymphocyteswereactivatedmorepronounced
when incubated with NSC631570 than with phytohemagglutinin. In rats NSC 631570
causedaclearincreaseofmacrophageswithNKͲactivity(49).
It was revealed in the tests on CBA mice and Wistar rats that NSC 631570 stimulates
macrophages. As marker of this activity the enzyme chitotriosidase, a part of the native
immunitywasused(168).


9
80

70

60
Cytolytic activity, %
50

40 SL
PL
30

20

10

0
0 0,59 1,18 2,36 4,72 9,45 18,9 37,8
µM Ukrain


Fig.4.TheeffectofNSC631570onthecytolyticactivityofspleen(SL)andperitoneal(PL)lymphocytes
fromalloimmunizedmice.FromLiepinsetal,1992(17).



80

70

60
Cytolytic activity, %

50
OU
40
MU
30

20

10

0
6 12 18 26 34
Days after alloimmunisation

Fig.5.Theeffectof1.18µMNSC631570onthecytolyticactivityofspleencellsharvestedatvarious
timeintervalsafterautoimmunization.FromLiepinsetal,1992(17).

In a study on intact and thymusͲectomised mice NSC 631570 augmented the endocrine
functionofthymus.AfterNSC631570administration,increasedproductionofsubstanceswith
thymosinͲlikeactivitywasdetected.RepeatedadministrationofNSC631570causeda2fold
riseofTͲcellsinblood,a4.5foldriseoflargegranulocytesandincreaseoftheNKͲactivityof
splenocytes.Theproductionofinterferonandantibodiesaftertheantigenadministrationwas
increasedaswell(180).
The effect of wellͲknown immune modulators interferonͲgamma, NSC 631570 and
pokeweedmitogenontheselectiveuptakeoftechnetiumͲ99m(99mTc)Ͳlabelledtumornecrosis
factor (TNF) was studied in the intramuscular implanted murine embryonic carcinoma. The

10
highest absolute tumor uptake of 99mTcͲ TNF was achieved when NSC 631570 was used,
followedbyIFNͲɶandpokeweedmitogen(fig.6;104).
In the experiments on BALB/c and F1 (BALB/c x C57BL/6J) mice it was revealed that NSC
631570 inhibits the allergic sensitization of animals against ovalbumin, expressed in the
weakened IgEͲreaction and decreased histamine release. The incubation of ovalbumin with
NSC631570induceddecreasedantigenicityofthisprotein(84).


Pokeweed mitogen

Ukrain

Interferon-gamma

Control

0 0,5 1 1,5 2 2,5 3 3,5



Fig. 6. Absolute (% administered dose/g) tumor uptake of ͲTNFͲɲ in mice BALB/c 1,5 h after
administration.AfterThakuretal,1992(104).

The immune modulating effect of NSC 631570 was studied in several studies in mice.
RepeatedsubcutaneousinjectionsofNSC631570tomiceinfectedwiththetwofoldLD50ofE.
coli,S.aureus,orinfluenzavirusincreasedthesurvivalrateoftheanimalssignificantly(60,87,
89).
Whenhumanlymphocyteswereincubatedwithphytohemagglutinin(PHA)andNSC631570,
increased absorption of 3HͲthymidin in the cells was observed. The authors point out the
strongsynergeticeffectofNSC631570andphytohemagglutinin(76).
BymeansofthecellproliferationassaythemitogeniceffectsofPHAandNSC631570on
humanperipheralbloodmononuclearcells(PBMC)werestudied.Itwasrevealedevenashort
pretreatment of the PBMC with NSC 631570 has a strong synergetic effect on the PHAͲ
mitogenesis.Consequently,thecellstimulationparametersweremuchhigheraftercombined
stimulationthanafterusingPHAalone(65).
Inexperimentswithmurine(CC57Black/6)macrophagesandrabbitGͲactin,theeffectsofNSC
631570andsanguinarineonphagosomeͲlysosomemembranefusionandactincytoskeleton
were studied. The most stimulating effect on the phagosomeͲlysosome fusion exerted
sanguinarineat10µmolandNSC631570at5µmol.AtthesamedoseNSC631570doubled
thecontentoffibrillaryactininmurineperitonealmacrophages.Moreover,NSC631570and
sanguinarine induced the polymerisation of rabbit globular actin. These effects were doseͲ
dependent. The authors suggest sanguinarine and NSC 631570 can alter intracellular
membranetransport(231).

11
The publications mentioned here point out that NSC631570 due to its many beneficial
immunepropertiescanbecategorizedasoneofthebestimmunemodifier.


Theangiogenicproperties

Another important feature of NSC631570 is the inhibition of the formation of the new
blood vessels supplying a tumor. Due to these antiangiogenic properties NSC631570
administered before surgery brings about better demarcation of the tumor from
surrounding tissue and the tumor encapsulation. This alleviates the surgical removal of
tumorswhathasbeenconfirmedinbreastcancerstudies(68Ͳ73,114).Intestsinvitro,
NSC631570inhibitedinadoseͲdependentmannertheproliferationofhumanendothelial
cells without exerting cytotoxic effect. The angiogenesis inhibition was observed on the
capillary formation model (136). This inhibition of the neoangiogenesis prevents the
metastasisformationaswell.


1600

1400
Total capillar tube length, µm

1200

1000 Kontrolle
10 µM Ukrain
800
20 µM Ukrain
600 50 µM Ukrain

400

200

0
1 2

Fig. 7. The length of the total capillary tubes after the incubation with NSC631570 in given
concentrations. 1 – HUVEC were incubated with NSC631570 for 4 h. 2 – HUVEC were
preincubated with NSC631570 for 4h and then incubated for 2 h in fresh medium without
NSC631570.HUVEC–humanumbilicalveinendothelialcells.AfterKoshelnicketal,1998(136).


12

I.EFFICACY

UKRAIN(NSC631570)ischelidoniiradixspecialliquidextract.Thisisacomplexproduced
fromtwoapprovedsubstances–greatercelandinealkaloidsandthiotepa(2,9,145).Its
qualityproofisspecifiedinGermanPharmacopoeaandPharmacopoeaAustriaca.

Ukrain is the first and only drug effective against cancer and more than 300 times less
toxic than its sources substances (12, 39, 41, 59, 111, 141, 179). It has been proven in
numerousinvitro,invivoandclinicalstudiesthatthismedicineinsmallerdosage(5mg)
exertsimmunemodulatingproperties(2,5,8,10,14)whereasinlargerdosageitseffect
ismalignocytolytic(3,6,11).ThetherapeuticindexofUkrainis1250(therapeuticindexis
relation between toxic dose and the therapeutic one and reflects the safety of a drug).
This is rather unusual for an anticancer drug and explains the good tolerability of NSC
631570. Therapeutic index of the common cytostatic drugs is in the range 1.4Ͳ1.8 and
theiroverdosagecancausefatalconsequences.


PhaseIClinicalStudy

The phase I clinical study was performed on 19 healthy volunteers on the outͲpatient
basis. Beside general clinical condition, following parameters were evaluated: blood
count, clinical chemistry, immune values, electrolytes, microelements, neopterin. NSC
631570wasadministeredintramuscularlyorintravenouslydaily,onthealternatedaysor
everythirddayatadailydoseoffrom5upto50mgfor7Ͳ40days.Inaspecialcase,the
medicine was administered during three years at a total dose of 3500 mg divided into
several therapy courses. No significant changes in clinical status were revealed at the
examination.Inthecaseofintramuscularadministration,volunteersreportedlocalpain,
sometimessleepiness,increasedthirstandpolyuria.Insomecases,alightnonͲsignificant
increaseofthebodytemperatureandminorbloodpressuredecreasewereobserved.The
authorsconcludedNSC631570atsingledosesof5,10,20and50mgwerewelltolerated,
alsoatprolongedadministration.(37)


TheDoseFindingStudy(PhaseII)

TofindoutthecorrectdosageforNSC631570aphaseIIclinicalstudywasperformedon
70 end stage cancer patients. The following parameters were estimated: physiologic
values (heart beat rate, blood pressure, body temperature), blood count, clinical
chemistry, electrolytes, immune values. The response on the therapy was evaluated by
means of xͲray, ultrasonography and computed tomography (CT). NSC 631570 was
administered intramuscularly or intravenously daily, on the alternate days, every third,
everyforth,oreveryfifthday.Singledoseswere2.5,5,10,15,20,or25mginascending
order(from2.5upto25mg),descendingfrom25mgto2.5mg,or5,10,15,20or25mg
constantly. The duration of a therapy courses was 10Ͳ90 days. Breaks between courses
variedfromsevendaysuptothreemonths.InallcasesthetherapywithNSC631579was
well tolerated. In some patients the analgesics dosage could be reduced. The quality of

13
lifeimprovedinthemostcases.Subjectiveaswellasobjectivesymptomsandsignswere
observed like headache, dizziness, thirst, sweating, polyuria, fever (with the body
temperatureincreaseof1Ͳ2°C),andpainatthetumorand/ormetastasesarea.Increased
temperatureatthetumorareawasobservedalso.Temporarytumorswelling,increased
heartbeatrateandminorbloodpressuredecreasewereobservedaswell.Theintensity
of such concomitants correlated with the response to the therapy. After full remission
theseconcomitantswerenotanymoreobserved(21,45).
In healthy volunteers, such concomitant events are observed not so extensively or not
observedatall.Itcanbesuggestedtheyaretriggeredbythetumordegradationproducts.
Theintensityoftheseconcomitantscanbedecreasedwiththedetoxicationmeasures.

According to the recent findings, to achieve the best results high doses of NSC 631570
shouldbeusedinturnwithsmallones.Higherdosesdestroytumorsandsmallerdoses
helps to eliminate the tumor degradation products. This is why alternate doses of NSC
631570 are used, e.g. 5Ͳ20 mg, or 5Ͳ30 mg, 5Ͳ40 mg daily or on alternate days. NSC
631570canbedilutedwith5%dextrose.Inthecaseofadministering20gNSC631570,
higherdosesofvitaminC(2Ͳ4g)shouldprecedetheinjection.Therearereportsontumor
responsesafter10dayinͲpatienttherapycourseswithdailydoseof20mgNSC631570
intravenously. Due to its antiangiogenic effect NSC 631570 can bring about the tumor
encapsulationimprovingitsrespectability.

TheclinicalefficacyofNSC631570hasbeenprovenbymanyresearchersandissubjectof
numerouspublications.Morethan40originalarticleskeeprecordsonthetreatmentof
morethan750patients,332oftheseweretreatedwithNSC631570incontrolledclinical
trials.AllresearchersnotetheefficacyandsafetyofNSC631570.


PhaseIIIClinicalTrials

Ukrain can cause the full regression of the main tumour and also of metastases. In the
treatment of advanced tumours Ukrain can improve the quality of life and prolong
survival.Manyclinicalstudieshaveprovedthis,suchasthoseoftheworkgroupsledby
Prof.BegerinGermanyandofProf.ZemskovinUkrainewithpancreaticcancer(182,186,
187,205,247;154,185),aswellasgroupsledbyProf.SusakandProf.BondarinUkraine
with colon cancer (67, 106, 108, 112). Neoadjuvant (before surgery) use of Ukrain can
induce encapsulation of tumours as revealed the studies by the researchers of Grodno
MedicalUniversity(Grodno,Belarus)inbreastcancer(68Ͳ73,114,157Ͳ159).

Inanopenstudytotal203advancedcancerpatientsweretreatedwithNSC631570and
partiallywithlocalhyperthermia(37.4%)afterallconventionaltreatmentmodalitieshad
failed and the disease progressed or relapsed. Full remission was achieved in 41 cases
(20.2%), partial remission – in 122 cases (60.1%). Seminoma and prostate cancer
respondedespeciallywellwithremissionrateofmorethan75%(144,161).


14

Pancreaticcarcinoma

In a controlled randomised study by Prof. Beger et al. in the Ulm University Hospital,
Germany,thetherapywithNSC631570andgemcitabinedoubledthesurvivalrateinthe
patients with inoperable advanced pancreatic cancer (182). The longest survival was 19
monthsinthegrouptreatedwithgemcitabinealone,26monthsinthecombinedgroup,
andintheNSC631570alonegrouptwopatientswerealiveafter28months.NSC631570
was well tolerated. The study authors consider further evaluation of NSC 631570 as
justifiedwhereasthequalityoflifeofthepatientsimproved(186).


Ukraininpancreascancer:palliativetherapy

Survival analysis in pancreatic cancer patients

0,9 Gemcitabine
Ukrain
Gemcitabine+Ukrain
0,8
Survival probability (Kaplan-Meier)

0,7

0,6

0,5

0,4

0,3

0,2

0,1

0
0 5 10 15 20 25 30

Time (months)

Gansauge,BegeretalLangenbeckcsArchivesofSurgery,2002

Patients were further observed after the conclusion of the study and it was noted that
UKRAIN was well tolerated and could be administered without problem to all patients.
UKRAIN brought about a significant increase in survival time in comparison to therapy
withgemcitabinealone.CombinationtherapywithgemcitabineandUKRAINshowedno
advantageovermonotherapywithUKRAIN.Thelongestsurvivalinthegemcitabinegroup
was19months,21monthsinthegemcitabine+Ukraingroup,andintheUkraingroupa
patientwasstillaliveafter28months.Theauthorsconcluded:‘Asaresultofthisstudy
we highly recommend the treatment of patients suffering from advanced pancreatic
cancerwithUkrain’(187).

2007theresultsofanotherclinicalstudybythesameresearchteamwerepublished.This
timetheefficacyoftheadjuvanttherapywithNSC631570hasbeendemonstratedinthe
patientswithadvancedpancreaticcanceraftersurgery.Thepatientsweretreatedwitha
combinationofNSC631570andgemcitabine.
The median survival was 33.8 months and the 5Ͳyear survival rate was 23.3% which is
clearly better than results reported in the earlier studieswithout NSC 631570, with the

15
median survival of 20.1 months and the 5Ͳyear survival rate was 21%
(http://content.nejm.org/cgi/content/abstract/350/12/1200).Moreover,NSC631570at
therapeuticdoserangehasonlyminimaladverseeffects,improvesthequalityoflifeof
patients and can be administered also on outͲpatient basis. All these features
distinguishesthisdrugfavourablecomparedtothestandardcytostaticagents.


Adjuvanttherapyinpancreascancer:comparisonofthreestudies

Author Neoptolemos Kurosaki Gansauge

Year 2001 2004 2007
Numberofpatients 238 16 30
NSC631570/
Therapy 5ͲFU/FS Gemcitabine
Gemcitabine
Relapsefreesurvival k.A. 16,8Mo. 26Mo.
Mediansurvival 19,7Mo. 20,4Mo. 37,6Mo.


Again, this publication supports the efficacy (and safety) of the use of Ukrain as it
demonstrates a considerable prolongation of survival compared to what is known from
otherclinicalstudies(247).

Other researcher confirmed the efficacy of NSC 631570 in pancreatic carcinoma (205,
208,209),whilethepartialremissionratewasashighas85.7%inonestudy(207).The
longestsurvivalinpalliativetherapywasmorethansixyears(185,186).

HistologicalchangescausedbythetreatmentwithNSC631570inthepancreatictumor
andinthesurroundingtissuewereprofoundlystudied.NSC631570hasbeenrevealedto
bringaboutthefibroticandsclerotictransformationofthetumour.Perivascularsclerosis
hasalsooccurred(206).


Colorectalcancer

In a controlled randomized clinical study by the National Medical University (Kyiv,
Ukraine)coloncancerpatientsweretreatedwithNSC631570orwith5ͲfuorouracilandxͲ
raytherapy.Thesurvivalrateafter21monthswas78.6%intheNSC631570groupand
33.3%inthegrouptreatedwith5ͲFUandradiotherapy(67).

WithinarandomizedstudyintheDoneckRegionalCancerCenter(Ukraine)rectalcancer
patientsreceivedeitherhighͲdoseradiotherapyand5ͲFUbeforesurgery,orthetherapy
withNSC631570:onecoursebeforesurgery(10mgeveryseconddayupto60mg)and
anothercourseafterwards(upto40mg).Duringfollowing14months,relapsesoccurred
insixpatients(25%)fromthecombinedgroupandin2patients(8.3%)intheNSC631570
group.Twoyearrelapseratewas33.3%(8patients)inthecombinedgroupand16.7%(4
patients)intheNSC631570group(112).Now,11yearsafterthispublication18from24
patients(75%)intheNSC631570grouparestillalive.

16

Prostatecancer

TheefficacyofNSC631570inprostatecancerhasbeenconfirmedinacontrolledclinical
study.Inthestudypatients,allstandardtreatmentmodalitieshadbeenexhausted.The
cancer relapsed and/or progressed and no therapy protocol was available. The patients
were treated with NSC 631570 and partially with local hyperthermia. Following results
wereachieved:fullremissionin54patients(73%),partialremissionin16patients(22%).
Onlyin4patients(5%)thetherapydidnotaffectthecourseofthedisease(201).


Totalnumberof Disease
Fullremission Partialremission
patients progression

74 54 16 4

100% 73% 22% 5%



ThegoodefficacyofNSC631570inprostatecancerhasbeenconfirmedinanotherstudy
(155).


Breastcancer

InacontrolledclinicalstudyconductedattheUniversityGrodno(Grodno,Belarus),after
thetherapywithNSC631570thehardeningofthetumor,aslightincreaseinthetumor
size (5Ͳ10%) and proliferation of connective tissues were observed. The T4/T8
lymphocytesratioincreasedby30%.Thetumoursappearedharderandslightlyenlarged
after NSC 631570 therapy, and were easier to detect by ultrasound or radiological
examination. Metastatic lymph nodes were also hardened and sclerotic (fibrous).
Tumours and metastatic lymph nodes were clearly demarcated fromhealthy tissue and
therefore easier to remove. Complications such as prolonged lymphorrhoea (leakage of
lymph onto the skin surface), skin necrosis (death of skin tissue), suppuration of the
wound,andpneumonia,alloccurredinpatientsfromthetwoNSC631570groupsatonly
halftheratethattheyappearedinpatientsfromthecontrolgroup.Basedontheresults
of this study the scientists from Grodno recommended the use of NSC 631570, at the
higher dosage, in all breast cancer operations (54, 68Ͳ70, 114). Other parameters were
also evaluated, e.g. hormones (T3, T4, cortisol, progesterone, estradiol, prolactin; 71),
immune values (lymphocytes, immune globulins, complement, phagocytic activity; 72),
morphologic and cytochemical changes (73, 110), amino acids and their derivates in
plasma(74,109)andinthetumortissue(75).

Inaseries ofarticlestheresearchershavestudiedtheeffectofNSC631570on various
parameters in breast cancer patients (157Ͳ160). Best results were achieved with higher
dosageofNSC631570.AlmosteverypatientnotedtheimprovementofthegeneralwellͲ

17
being,sleepandappetite.Duringthesurgery,thetumorsaswellasinvolvedlymphnodes
were presented sclerotic and well demarcated from the surrounding tissue. This
alleviated the surgical removal of the tumor considerably (158). In the tumor tissue,
increased concentration of the amino acid proline was revealed indicating augmented
production of connective tissue that demarcates the tumor from surrounding tissue
(159).NSC631570improvedalsotheaminoacidbalanceofpatients(160).


Bladdercancer

InastudyNSC631570causedfullremissioninthreepatientsforsixmonths(113,137).

Biochemical evaluation revealed NSC 631570 had favourably affected the amino acid
metabolism(156).


Malignantmelanoma

ThefirstpublicationontheusingNSC631570inmalignantmelanomadescribesthefull
remissioninapatientwithmetastasestothelung(91).

Alonglastingremission(morethan10yearswithoutrecurrence)hasbeenobservedina
patientwithmalignantnodularmelanomaafterthetreatmentwithNSC631570.Atthe
beginning of the NSC 631570 therapy liver metastases were present and melanin was
excretedwithurine(92).

The effects of NSC 631570 alone and in combination with the pathogen associated
molecules (PAM) on the cell cycle and apoptotic induction were compared in two
melanoma cell lines MMͲ4 and MMͲ4M2 with different metastatic properties (cell
divisionrate,hematogenousmetastazing,sensitivitytotheTNFͲinducedapoptosis).
Apoptosis induction and cell viability were analyzed using trypan blue exclusion test,
morphological criteria, DNA gel electrophoresis, and flow cytometry. Cell cycle
distribution of tumor cells was estimated by flow cytometry. The therapy with NSC
631570inducedapoptosisinbothmelanomacelllinesinadoseͲdependantmatter.The
cell line with higher metastatic potential was more sensitive to NSC 631570. In the cell
line with low metastatic potential, combined use of NSC 631570 and PAM induced
apoptosismoreeffectively(261).


Braintumors

NSC631570hasbeensuccessfullyusedinthetreatmentofbraintumors(101,102).

InareviewontheclinicalstudieswithNSC631570performedsofartheresearchersfrom
the Universities of Exeter & Plymouth suggested this agent to have potential as an
anticancerdrug(238).


18
Malignantgynaecologictumors

Earlier,therewasareportonthesuccessfulusingNSC631570inthetreatmentofovarian
cancer(97).AlsointhetestsofNationalCancerInstitute(Bethesda,Maryland,USA)NSC
631570wastoxicagainstallovariancancercelllinestested(190).Otherauthorsreported
ongoodresultsinthetherapyofcervicalcancer(27,96).


Ukrain(NSC631570):autofluorescenceunderUVͲlight




In ultraviolet light Ukrain fluoresces in the yellowish green range of spectrum. Excitation
frequenciesarewithinarangeof220to490nm.Thespectralwidthofthefluorescenceextends
from410to665nm(4).





ThinͲlayer chromatography plate with drops of Ukrain under UV light. On the left, Ukrain in a
concentrationof10mg/mlindistilledwater.Thenserialdilutionsbyafactorof10eachtime(1
mg/ml;0.1mg/ml,…).



19


Patient N.E., aged 82. 1½ year history of multiple and exulcerating basaliomas at the
cheekͲnosearea.




The same patient under UVͲlight at 254 nm three minutes after the first intramuscular
injection of 5 ml Ukrain. Strong fluorescence of the tumours and surrounding tissue is
visible. Ukrain administered after one week produced only slight fluorescence. A
considerableregressionofthetumourswasalsoobserved.

20
The selective accumulation of NSC 631570 in the tumor tissue proven due to
autofluorescence

The selective effect of NSC 631570 on cancer cells has been confirmed due to its
autofluorescenceunderUVlight(4).FirsttimethisfeatureofNSC631570waspresented
1983at13thInternationalCongressofChemotherapyinVienna.Ithasbeenconfirmedin
thisworkNSC631570toselectivelyaccumulateincancercells.TheaccumulationofNSC
631570incancercellscorrelateswiththeefficacyofthedrug.Withtheeliminationofthe
preparationfromthebodytheintensityofthefluorescencedecreasesalso(1).
Atthiscongress,thefirstreportsonthesuccessfulusingNSC631570inthetreatmentof
theendͲstagecancerpatientswerepresented,whereallstandardtherapymodalitieshad
failed.Thoughpoorprognosis,NSC631570broughtaboutfullremissioninapartofthe
patientsandsomeofthosearestillalive.



CASEREPORTS

Besides clinical studies, NSC 631570 has been also used by many physicians in the
treatment of various tumors, for example cervical cancer (96), ovarian cancer (97),
testicular cancer (98), esophageal carcinoma (99), urethral carcinoma (100), and
neuroblastoma(116).SuchreportscompletethepatternoftheclinicalusingNSC631570.


Breastcancer

Physicians report on the successful treatment of a stage IV breast cancer patient. After
theNSC631570therapythetumorsizedecreasedandthesurgicalremovalwasalleviated
(162).

There are also more reports on the successful treatment of breast cancer (94, 95),
includingarelapsingbreastcarcinomawithlungmetastases(94).


Sarcomas

Beside common solid cancers, infrequent sarcomas present a real problem in oncology
becauseoftherapyresistance(93).Allthemoreinterestingisthecaseofretroperitoneal
sarcoma successfully treated with NSC 631570. At the time of the publication, e.g. four
yearsafterthestartoftheNSC631570therapy,thepatientwasinfullremission(163).

TheEwing’ssarcomaisatumorfromconnectivetissuecellsofbonemarrow.Itisarare
diseasewiththeincidenceof3newcasespermillion.Ewingsarcomacanoccuronevery
siteofskeleton.Inmostcases,legs,pelvicbone,scapulaandribsareconcerned.Usually,
children between 10Ͳ15 years suffer from it, but also children under 10 years can be
affected.


21
A 9 yearͲold girl from Poland was diagnosed with Ewing's sarcoma. The girl received
chemotherapy and radiotherapy but the tumour growth could not be stopped. The girl
wasdeclaredtohaveexhaustedallpossibleformsoftreatment(i.e.hadbeengivenupby
mainstreammedicine)andwassenthomewithinfaustprospects.Theparentstooktheir
daughtertoViennatoSt.AnnaChildrenHospitalbecausetheyhopedtoreceivethestateͲ
ofͲtheͲarttreatment.Thedoctorscarriedoutnewexaminationsandhadtodeclarethat
theytoocouldnothelpher.Thecompleterepertoireofmainstreammedicinewasused
up but the tumour growth continued. At this point her parents heard about Ukrain by
chanceandcontactedDr.WassilNowicky.On21January1984treatmentwithUkrainwas
begun.
After six months of treatment with Ukrain the girl was examined again at St. Anna
Children'sHospitalandtothegreatastonishmentofthedoctorsnotonlyhadthetumour
growthbeenstoppedbutevenbetter,thetumourhadbecomesmaller.However,there
wasstillnointerestinUkrain.
Treatment with Ukrain was continued and every six months the progression of the
diseasewascheckedatSt.AnnaChildren'sHospitalattheinstigationofDr.Nowicky.In
thiswayDr.NowickywantedtoarousetheinterestofdoctorsinUkrain(unfortunatelyto
noavail).
GraduallythetumourdisappearedcompletelyandattheXͲrayexaminationonOctober
31, 1990 it was seen that even the bone which had been eaten away had regenerated
(28).

Ewing’s sarcoma, first diagnosed 22.11.1983, histologically verified, tumour resistant
bothtochemotherapyandradiotherapy.UKRAINtherapystartedon21January1984.



A 9 yearͲold girl had felt marked pain below the right knee joint in November 1983
following a slight injury. XͲray revealed Ewing’s sarcoma in the proximal portion of the
right fibula. Hospital treatment included chemotherapy and cobalt therapy. XͲrays
confirmedthatthepatient’stumourhadnotrespondedtoradiationorchemotherapyand
the tumour mass increased rapidly. One month after the end of chemotherapy, UKRAIN
treatmentwasstartedatadoseof5mgi.m.foratotalof10injections,combinedwith
regional deep hyperthermia. The first series of UKRAIN therapy included three identical
courses with a twoͲweek pause between them. Six series of UKRAIN treatment were
administered over the course of one year. Repeated xͲrays showed reduction of the
tumourmass.


22

In a 10 year girl the Ewing’s was verified histologically. She was treated in the high risk
groupoftheEICESS92study.TheMRIofthepelvisrevealedthediseaseprogression;the
tumor was resistant to chemotherapy and to radiotherapy as well. The treatment with
NSC 631570 was started: 15 mg mixed with 250 ml dextrose 5% and 5 g vitamin C
followed by local hyperthermia on alternate days, 10 sessions totally. The control MRI
revealed the stop of the progression. The followed therapy cycles brought about the
regressionofthetumor.AttheMRIafterfouryearsnosignsofarelapseormetastases
couldberevealed(115).Thepatientisinremissiontillnow.



Ewing’s sarcoma, first diagnosed 18.3.1996; the tumour was resistant to both
chemotherapyandradiotherapy.UKRAINtherapystartedon13October1997(115).



The patient, a 10 yearͲold girl, was treated in the highͲrisk arm of the EICESS 92 study. MRI
examinationofthepelvicregionon1.9.1997showedprogressioninthecysticͲedematousprocess.
She was then treated with combined Ukrain and local hyperthermia therapy. The therapy series
consistedof15mgUkraininaninfusionwith250mlglucoseand5gvitaminC,followedbylocal
hyperthermia treatment. Treatment was administered every second day up to a total of 10
therapysessions.MRIexaminationon8.1.1998showednoprogressionofthetumour.Subsequent
therapy cycles caused regression of the tumour (see MRI on 15.6.1999 and 1.2.2000). MRI on
1.2.2001:Cysticresidualdefectinrightfemur,asobservedinpreviousexaminations.Nosignofa
relapseorofmetastases.



Invitrostudies(studiesoncellcultures)byscientistsattheUniversityofTübingenhave
demonstrated the effectiveness of Ukrain with Ewing's sarcoma. These studies and
clinicalsuccesses,suchaswithDr.Aschhoff,wererepeatedlysubmittedtotheMinistryof
Health.AtDr.Aschhoff'sclinic‘Cancerpatientsweretreatedwhosediseasehadalready
beentreatedwithallmainstreammedicinetherapyprotocolsandasaresultofrelapse
and/or progress of the disease no further mode of therapy was available for them and
who had thus exhausted all modes of therapy’ (161). In these patients, Dr. Aschhoff
achievedhighremissionrateusingNSC631570,forexample,50%forEwing'ssarcoma.


23
Renalcellcarcinoma

NSC631570hasbeenusedforthetreatmentofrenalcellcancerafterthetherapywith
vinblastinhadfailedandthetumorhadspreadmetastases.Fullremissionwasachieved
withUkraintherapyandlasted32monthsatthetimeofpublication(164).


Testicularcancer

NSC 631570 has been used in the combined treatment of a nonseminoma patient and
improvedhisimmuneparameters(98).


Esophaguscarcinoma

The therapy of an esophagus carcinoma patient with NSC 631570 induced a prolonged
remission(99).


Bladdercancer(urothelialcellcarcinoma)

Acaseofasuccessfultreatmentofurothelialcellcarcinomawasdescribed.Theauthors
reportedonasustainedremissionandimprovedqualityoflifeofthepatient(100).


Neuroblastoma

After chemotherapie had failed, the patient was treated with NSC 631570. The tumor
markersdecreased,themetastasesrespondedwellonthetherapyandthequalityoflife
ofthepatientimproved(116).


Hereditarydiseases

Tuberoussclerosis

As an example of the using NSC 631570 in hereditary diseases, a case of a girl with
tuberous sclerosis can be considered. After successful treatment the patient is now an
adultwomanandhasgivenbirthtoahealthyson(210).


Generalisedlymphangiomatosis

Another publication reports on the successful treatment of a severe hereditary disease
generalisedlymphangiomatosis(211).ThethreeyearͲoldStefanDanwiththediagnosisof
generalisedlymphangiomatosiswassenthomebydoctorsin1995ashavingexhaustedall
forms of therapy. To “comfort” his parents they were told that Stefan would never be

24
able to speak and walk in his life. After two years of UKRAIN therapy with a general
practitioner the child was most certainly able to speak and also to walk. However,
treatmentwithUKRAINwasstopped.Thetumourthenbegantogrowandbroughtabout
acompressionofthespinalcord.Stefan–inthemeantimeeightyearsͲold–underwent
surgery.Despitethesurgicalinterventiontheconditionoftheyoungpatientdeteriorated
continually, resulting in him having to be connected to a lung ventilator. Paraplegia
appeared.Inthisconditionthechildwasdischargedtobecaredforathome.Hehadto
be given morphine four times a day to relieve his pain and his breathing had to be
supportedwithahomerespirator.Thedoctorsthenafterallrecommendedhisparentsto
turn to UKRAIN therapy again. Although the child’s condition could be improved with
UKRAINtherapy,Stefancanspeak,read,isveryintelligent,butwillnolongerbeableto
walk.


Xerodermapigmentosum

Xerodermapigmentosum(XP)isageneticdisorderofDNArepairinwhichtheabilityto
repairdamagecausedbyultraviolet(UV)lightisdeficient.Multiplebasalcellcarcinomas
(basaliomas) and otherskin malignancies frequently occurat ayoung age in those with
XP. In fact, metastatic malignant melanoma and squamous cell carcinoma are the two
mostcommoncausesofdeathinXPvictims.

Thisisaveryraredisease.Theincidencediffersregionallyandisbetween1:40000(Japan)
and1:250000(USA).About250XPpatientsliveintheUSA,about50inGermany,mostly
children.Thelifeexpectanceislow;usuallytheydieinthefirstdecade.Ifleftunchecked,
damagecausedbyUVlightcancausemutationsinindividualcellsDNA.XPpatientsareat
a high risk (more than 2000 times over the general population) for developing skin
cancers,suchasbasalcellcarcinoma,forthisreason.
AreportonthesuccessfulusingNSC631570inaXPpatientsuggeststhisdrugcanbevery
usefulalsointhishereditarydisease(212).




25
Ukrain(NSCͲ631570)beiXerodermapigmentosum

PatientS.S.,aneightyearoldboy,waspresentedwithanulceringlesionofthenose.Ashe
was10monthold,xerodermapigmentosumwasdiagnosed.Untiltheageofthreeyears
thenumberofskinlesionsincreasedconsiderably.InMay2002skincancer(squamouscell
carcinoma)atthenosewasdiagnosed,T4NXM0,histologicallyverified.FromMaytillJune
2002threecyclesofchemotherapywereadministered(cyclophosphamide,vincristine,and
vinblastine). The therapy failed and the tumors grew up. Clinical investigation in April
2004revealeddeformingmalignantmelanomaofthenosewithinvasionintothecartilage
ofnasalseptum,measuring3x3cm.On20May2004thetherapywithUkrainwasstarted,
5mgintravenouslytwiceaweek,uptoatotaldoseof85mg.Onemonthafterthelast
administrationofUkrainacompleteregressionofthetumorwasrevealed.Theskindefect
was partially replaced with connective tissue. Xeroderma skin lesions improved
throughoutthebody.

  
  
Photo1.PatientS.S.beforethe Photo2.Autofluorescenceof Photo3.PatientS.S.in
therapywithUkrain.Deforming NSCͲ631570atthemelanoma December2004.Complete
invasivemalignantmelanoma areaunderUVͲlightduringthe regressionofthetumor,with
ofthenose.April2004. firstintravenousinjection.May connectivetissuesubstitution.
2004.



26

ANTIVIRALPROPERTIES

The researchers from St. Petersburg, Russia have used the antiviral properties of NSC
631570 in their works with viral hepatitis C (HVC). They reveal NSC 631570, at optimal
dosage,hasbroughtabouttheeliminationofthevirusfromthebloodin40(80%)of56
patients(165).

In their further study these researcherscompared the effects of NSC631570 at various
dosageswiththerecombinanthumaninterferonͲalphaͲ2b(IFN)in75HVCpatients.The
bestresultswereachievedwhenNSC631570wasadministeredatasingledoseof1mg
(203).

PilotstudiesevaluatingtheeffectofNSC631570onHIVvirusandrelateddiseaseswere
alsoperformed(12).Theauthorsnoted,forexample,improvedimmunevaluesafterthe
therapy(103).

TheantiviralpropertiesofNSC631570wereconfirmedintheinvivoexperiments(42,51,
52,88,90).





THEINHIBITIONOFTHETUMORALANGIOGENESIS

NSC 631570 inhibits the formation of the new blood vessels supplying a tumor. Due to
these antiangiogenic properties NSC 631570 administered before surgery brings about
betterdemarcationofthetumorfromsurroundingtissueandthetumorencapsulation.
Thisalleviatesthesurgicalremovaloftumorswhathasbeenconfirmedinbreastcancer
studies(68Ͳ73,114).Itisrecommendedtoreducethetumorburden7Ͳ10daysafterthe
startofthetherapywithNSC631570.

In tests in vitro, NSC 631570 inhibited doseͲdependent the proliferation of human
endothelial cells without exerting cytotoxic effect. The angiogenesis inhibition was
observed on the capillary formation model. The tumor angiogenesis is the formation of
new blood vessels supplying a growing tumor with nutrients. Angiogenesis is of critical
importanceforthetumorgrowth(136).


27
InhibitionofthecapillarysproutingbyNSC631570intheendothelialspheroidsmodel





Pancreatic adenocarcinoma, preoperative treatment with Ukrain, total 100 mg, 10 days
prior to surgery (pancreaticoͲduodenectomy). Formation of a capsule (A) around the
tumour.Tumourcellsdonotinfiltratethecapsule.Massiveroundcellinfiltration(B)ofthe
tumour–capsuleborderarea.HͲe,x100.







28
Pancreatic adenocarcinoma, preoperative treatment with Ukrain, total 100 mg, 10 days
priortosurgery.Necrosisoftumourtissue(A),inhibitionofnewvesselsformation(defect
incapillarywall,defectinendothelialcovering,B).HͲe,x200.






Pancreatic adenocarcinoma, preoperative treatment with Ukrain, total 100 mg, 10 days
prior to surgery. Nuclear changes: Chromatin dispersion (A) and fragmentation (B),
hydropiccytoplasmdegeneration(C).“Iron”hematoxylin–vanGieson,x500.






29
THEINVITROSTUDIES

Clinical efficacy of NSC 631570 is not coincidental or even ‘spontaneous remission’ but
ratheraconsequenceofitsmechanismsofactionconfirmedinvariousinvitroandinvivo
studies. NSC 631570 has been tested on more than 100 cancer cell lines so far.Among
others, NSC 631570 was tested at the National Cancer Institute (Bethesda, Maryland,
USA) on 60 cell lines representing eight important human malignant tumors: brain
tumors, ovarian, small cell and nonͲsmall lung cancer, colon cancer, kidney cancer,
leukaemia and malignant melanoma. NSC 631570 exerted toxic effectsagainst all these
cell lines (40, 190). Compared to 5Ͳfluorouracil (5ͲFU) and gemcitabine, two standard
cytotoxicagentsinthetreatmentofdigestivetracttumors,NSC631570achievedbetter
resultsandnotonlyinhibitedthecellgrowthbutreducedthecellmass,also.


ThecytotoxiceffectofNSC631570onthecancercelllinesMͲHeLaandHepͲ2/0Ͳ6Ͳ5war
morepronouncedthantheeffectofasyntheticagentOliphen(61).NSC631570inhibited
the growth of four Ewing’s sarcoma cell lines doseͲ and timeͲdependent. In this
experimentthe effect of NSC 631570 was more pronounced than this of thiotepa (243,
discussedin244).InthetestsonEhrlich’scarcinomacellsandlympholeucemiaPͲ388,the
researchersrevealedthatthesensitivityofthecancercellstoNSC631570dependshighly
on the cell cycle phase, the first sensitivity peak being at the end of G1 phase and the
second Ͳ in G2 phase (148). The effects of glucose, succinat, pH value and increased
temperatureontheefficacyofNSC631570againstcancercellswereinvestigatedinvitro.
GlucosereducedthecytotoxiceffectofNSC631570,butsuccinatintensifiedit.Themost
pronouncedeffectwasatpH7.3Ͳ8.0.Thetemperatureupto41.5°Cdidnotimpactthe
effectofNSC631570(117).

30
Results of the Ukrain (NSC 631570) study at the National Cancer Institute, Bethesda,
Maryland,USA.TheeffectsofNSC631570onthe60varioushumancancercelllines
Malignant Melanoma Colon Cancer
100

Percentage Growth
Percentage Growth

100

50 50

0 0

-50 -50

-100 -100
0,005 0,05 0,5 5 50 0,005 0,05 0,5 5 50
UKRAIN concentration, µg/ml UKRAIN concentration, µg/ml

Ovarian Cancer Lung Cancer


Percentage Growth 100
100
Percentage Growth

50
50
0
0

-50 -50

-100 -100
0,005 0,05 0,5 5 50 0,005 0,05 0,5 5 50
UKRAIN concentration, µg/ml UKRAIN concentration, µg/ml

Renal Cancer Brain Cancer


Percentage Growth

100 100
Percentage Growth

50 50

0 0

-50 -50
-100 -100
0,005 0,05 0,5 5 50 0,005 0,05 0,5 5 50
UKRAIN concentration, µg/ml UKRAIN concentration, µg/ml

Normal Cells
Percentage Growth

100

50

-50

-100
0 10 20 50
UKRAIN concentration, µg/ml


31
THESELECTIVEEFFECTOFNSC631570


IncomparativestudiesNSC631570wastestedon18malignantand12nonͲmalignantcell
lines at identical conditions (36, 38, 63, 143, 147, 149, 181, 184, 190, 245, 255). These
experiments eliminated all doubts on the selective effect of NSC 631570. Numerous
studieshaveconfirmedNSC631570tobethefirstandonlyanticanceragentbeingtoxic
againstcancerbutnotnormalcells.Thisexplainsalsoitsgoodtolerabilityinclinicaluse.

First indications on the selective effect of NSC 631570 on the cancer cells provided an
earlystudyin1976oftheBundesstaatlichenAnstaltfürExperimentellͲPharmakologische
und Balneologische Untersuchungen (Vienna, Austria). This study revealed different
oxygen consumption by normal liver cells and Ehrlich’s tumor ascitic cells after the
incubationwithNSC631570wasrevealed(38).

About at the same time, the researchers from the Vienna University of Agriculture
compared the inhibiting effect of NSC 631570 on the proliferation of malignant and
normal cells. For 50% growth inhibition of normal endothelial cells, the NSC 631570
concentration had to be tenfold higher than for the same growth inhibition of human
osteosarcomacellline(36).

The different influences of Ukrain on oxygen consumption in healthy cells and cancer
cells


 
TheeffectofUkrainontheoxygenconsumption TheeffectofUkrainontheoxygenconsumption
in malignant cells (murine Ehrlich’s ascites in normal cells (sediment from guinea pig liver
tumoursuspension) homogenate)


32

TheuptakeofUkraininmelanomacellscomparedtonormalcells(invitro)

Phase Contrast Fluorescence

Melanoma cells High uptake

Endothelial cells Low uptake



HohenwarterO.etal.SelectiveinhibitionofinvitrocellgrowthbytheantiͲtumourdrug
Ukrain,DrugsunderExperimentalResearch,1992(36).


This selective effect of NSC 631570 on cancer cells has been confirmed in numerous
studiesattherenowneduniversitiesandresearchfacilities.

InthestudyfromEuropeanOrganisationforResearchandTreatmentofCancer(EORTC),
humantumorxenografts(HTX)wereimplantedintonudemice.Thesetumorcellswere
later incubated with NSC 631570 at various concentrations. Following HTX were used:
coloncancerCXF1103/11,stomachcancerGXF217/17,lungcancerLXFL529/14,breast
cancer MAXF 401/13, melanoma MEXF 276/10, and ovarian cancer OVXF 899/9. NSC
631570wasactiveagainstOVXF899/9at10µg/mlandinallcoloniestestedat100µg/ml
withtheT/CͲratio(‘testtocontrol’)1/135inOVXF,8/109inCXF,10/98inGXF,15/187in
LXFL,34/133inMAXF,and10/122inMEXF(64,189).

In1998,atthe89thAnnualMeetingoftheAmericanAssociationforCancerResearchin
New Orleans, USA, researchers from the University of Pretoria, South Africa presented
theirworkontheselectiveeffectofNSC631570onvariouscancercelllines.Theauthors
concluded‘thatUkrainisselectivelytoxictomalignantcellsbycausingametaphaseblock
which is characterised by abnormal chromosomal distribution, and results in the
formationofmicronucleiandinapoptosis’(139).In2000thesamegroupdiscoveredNSC
631570toinhibitthetubulinpolymerisation.Inthispapertheydeniedtheselectivemode
ofactionofNSC631570oncancercells(140).

ResearchersfromEberhardͲKarlsͲUniversityTubingen,Germany,investigatedtheeffects
of NSC 631570 on cell survival, alteration of the cell cycle and induction of apoptosis
without and in combination with ionising radiation (IR) at a dose of 1Ͳ10 Gy. The tests
wereperformedontheexponentiallygrowinghumantumorcellsMDAͲMBͲ231(breast),

33
PAͲTUͲ8902(pancreas),CCLͲ221(coloncancer),UͲ138MG(glioblastoma),andhumanskin
andlungfibroblastsHSF1,HSF2andCCD32ͲLU.WithoutIR,NSC631570exertedatimeͲ
and doseͲdependant cytotoxic effect, more pronounced against the cancer cells. Flow
cytometryrevealedNSC631570tomodulateradiationtoxicityagainsthumancancercell
linesandtoprotectnormalcellsfromradiation.ThecombinationofNSC631570plusIR
gave enhanced toxicity in CCLͲ221 and UͲ138MG cells with their accumulation in the
G2/M phase of the cell cycle, but not in MDAͲMBͲ231 and PAͲTUͲ8902 cells. A radio
protective effect was found in normal human skin and lung fibroblasts. The authors
suggestareasonableuseofNSC631570incombinedradiochemotherapy(184).

ThecytotoxiceffectsofNSC631570wereevaluatedintwoprimarypancreaticcancercell
lines (PPTCC), fibroblasts derived from pancreatic ductal adenocarcinoma specimens (FͲ
PDAC),andanimmortalizedepithelialductalpancreaticcellline(HPNE).Cytotoxicitywas
assessed by the CellTiter 96 kit based on the cellular metabolism of the tetrazolium
compoundXTT,whichisreducedbylivingcellstoyieldasolubleformazanproductinthe
presenceoftheelectroncouplingagentphenazinemethosulfate,whilethemodulationof
NSC 631570 uptake in the medium was studied using the fluorescence of NSC 631570
withtheAlphaDigiDocsoftwarebyUVlightexcitation.CytotoxiceffectsofNSC631570in
PPTCCsweresignificantlyhigherthanthoseobservedinFͲPDACandHPNEcells(20%vs.
80%alivecells).Furthermore,theULAͲDCtestrevealedthatPPTCCscellsconsumedmore
drugthanFͲPDACandHPNEcells.ThesedatademonstratedtheselectiveeffectofUkrain
inPPTCCs,whichmayberelatedtoadifferenttransportsystemorhighermetabolismof
thedruginPDAC,andwarrantfurtherinvestigationsinordertosupportthepossiblerole
ofUkraininPDACtreatment(265).



InductionoftheApoptosisinCancerCells

In the study on the erythroleucemia cells KͲ562, it was revealed NSC 631570 to bring
aboutthebimodaldeathofcancercells.AtlowerconcentrationsofNSC631570,cancer
cells die in as a consequence of apoptosis. At higher concentrations, the formation of
microtubulesisinhibitedandpolyploidyoccurs(56,62).

The researchers from the Rochester University, USA revealed NSC 631570 causes the
accumulationofprostatecancercellsaswellasepidermoidcarcinomacellsintheG2/M
phase,however,notofnormalcells(149,150).

In the tests on human cervix carcinoma cells HeLa, squamous carcinoma cells WHCO5,
normalkidneycelllineGraham293,andtransformedkidneycelllineVerofromAfrican
green monkey the researchers of the University of Pretoria, South Africa revealed NSC
631570 ‘is selectively toxic to malignant cells by causing a metaphase block which is
characterised by abnormal chromosomal distribution, and results in the formation of
micronucleiandinapoptosis’(139).

ThescientistsoftheEberhardKarlUniversity(Tübingen,Germany)investigatedtheeffect
ofNSC631570onthecellsurvival,thecellcyclemodificationandtheapoptosisinduction

34
aloneandcombinedwithradiation(IR).TheydiscoveredNSC631570combinedwithIR
increased the toxicity against the cell lines CCLͲ221 and UͲ138MG. The normal human
skinandlungfibroblastswereprotectedfromthedamagingeffectsofIR(184).




Ukrain’s(NSC631570)deadlyactionagainstcancercellsͲInductionofapoptosis

Apoptosis




Estimating the cell proliferation according to the BrdU uptake in the cell lines AsPC1,
BxPC3, MiaPaCa2, Jurkat, and THPͲ1 and the cell cycle phases by means of Giemsa
staining, the authors established NSC 631570 at a dose of 10 µg/ml brought about a
considerableaccumulationofcancercellsintheG2/Mphaseafter24hincubation.The
apoptosis rate in the peripheral mononuclears was similar at the same incubation
conditions.Moreover,themitogenestimulatedlymphocytesshowedincreasedblastogen
reaction(181).

TheapoptosisinductionbyNSC631570hasbeenalsoconfirmedintheinvitrotestson
the Chinese hamster ovarian cells. In this experiment the effects of NSC 631570 and
etoposideweresynergistic(167).



InhibitionoftheTubulinPolymerisation

In the experiments with cow brain tubulin, the scientists from the Pretoria Universioty
(SouthAfrica)discoveredNSC631570toinhibitthetubulinpolymerisation(146).


35
Lateron,theresearchersfromtheUniversityofUlmrevealedinthetestswithpancreas
cancer lines AsPC1, BxPC3, Capan1, MiaPaCa2, and Panc1 NSC 631570 brings about a
dosedependantcellcyclearrestintheG2/Mphase.Moreover,NSC631570stabilizesthe
tubulin monomers in cancer cells and consequently inhibits the formation of
microtubules(143).


ActivationofMitochondrialCaspases

Caspases, or cysteineͲaspartic proteases are a family of enzymes, which play essential
rolesinapoptosis(programmedcelldeath)henceincancertherapy.

InthetestsontheJurkatlymphomamodel,NSC631570hasbeenproventobeastrong
apoptosis inductor. Profound research showed NSC 631570 brought about the
depolarisationofmitochondrialmembranesandconsequentlytheactivationofcaspases
(246).

There are two types of apoptotic caspases: initiator (apical) caspases and effector
(executioner) caspases. Initiator caspases CASP8 and CASP10 induce the death factor
dependantorextrinsicapoptosispathway,whereaseffectorcaspaseCASP9mediatesthe
intrinsicormitochondrialapoptosis.InthetestsattheNacionaldeCancerologia,Mexico
City,MexicoNSC631570revealedthatUkraininducedapoptosisinapanelofcancercell
lines(cervicalcancerHeLa,HeKB,HeKS32,HeBcl3,HeNFRandHeIKK,humancoloncancer
SW480,humanrenalcarcinomaHEK293,humanosteosarcomaMGͲ63)byactivatingthe
intrinsic cell death pathway. Interestingly, nonͲtransformed fibroblasts hTERT were
insensitivetothedrug(255).


TheEffectonCyclinsandCyclinͲDependantKinases

Cyclins are a family of proteins which control the progression of cells through the cell
cyclebyactivatingcyclinͲdependentkinase(Cdk)enzymes.Acyclinformsacomplexwith
Cdk.ComplexformationresultsinactivationoftheCdkactivesite.Whenconcentrations
in the cell are low, cyclins dissociate from Cdk, thus inhibiting enzymatic activity; this
probably occurs due to a protein chain of the Cdk blocking the active site upon cyclin
dissociation.Thereareseveraldifferentcyclinswhichareactiveindifferentpartsofthe
cell cycle and which cause the Cdk to phosphorylate different substrates. Increasing
concentrationofcyclinAtriggersthecellintotheG2phase,whereascyclinBisessential
forthemitosisinitiation.

The researchers from the Rochester University explored the effect of NSC 631570 on
cyclins and Cdk in the epidermoid cancer cell lines ME180 and A431 as well as in the
prostatecarcinomalineLNCaP.Theyfoundchangesintheconcentrationsofmitoticcyclin
AandB1aswellasCdk1andCdk2.IncreasedexpressionofCdkinhibitorp27hasbeen
alsoobservedinbothcancercelllines.Thiscanpromoteaccumulationofthecellsinthe
G2/Mphase(147,149).


36

TheEffectontheExpressionofhENT1anddCK

The interactions between NSC 631570 and the molecular determinant expressions
involvedinthemetabolismofgemcitabine,suchashENT1anddCKwereevaluatedinin
vitrostudiesonpancreaticductaladenocarcinomacells(PDAC).TwoATCCcelllines(PL45
and MiaPaCaͲ2) and 2 Primary Cell Cultures obtained from PDAC patients underwent
surgicalresections(PPTCC78andPPTCC109)wereused.CellsweretreatedwithUkrainat
IC50 concentration levels for 48h. All the amplifications were carried out with
normalizationofgeneexpressionagainsttheglyceraldehyde3Ͳphosphatedehydrogenase
(GAPDH) housekeeping control gene, and the quantitation of gene expression was
performed.NSC631570positivelymodulatedtheexpressionofhENT1mRNAinallPDAC
cell cultures treated with IC50 (p<0.001). The analysis revealed a mean increase of 2.8
fold (p=0.001) compared to untreated control cells. In PL45 and MiaPaCaͲ2 cells NSC
631570positivelyaffectsmRNAexpressionofdCKgeneaswell(264).
BasedonthepreviousclinicaldatatheNSC631570Ͳgemcitabinecombinationappearsa
promisingregimenandtheresultsofthisstudyprovidedtheexperimentalbasisforthe
furtherclinicaltestingoftheNSC631570ͲgemcitabinescheduleinPDACpatients(Funel
et al, ‘Molecular mechanisms underlying the synergistic interaction of the novel
anticancerdrugUkrainwithgemcitabineinpreclinicalmodelsofpancreaticcancer’,44th
AnnualPancreasClubMeeting,NewOrleans,USA2010).


TheEffectontheDNAandProteinSynthesis

TheeffectofNSC631570ontheDNAandproteinsynthesisincancercellswasstudiedin
vitroonthecelllinesHeLa,Yoshida,micelymphoma,micemyeloma,humanWiDrtumor,EB,
EsB, YACͲ1, und P815. NSC 631570 (1Ͳ100 µg/ml) has been proven to inhibit in doseͲ
dependantwaytheDNA,RNAandproteinsynthesisinthesecelllines.Atthesametest
conditions,theinhibitioninnormalcells(humantonsilcellsandguineapiglivercells)was
muchlesserdistinct(58,63).

Among many possible mechanisms of action of NSC 631570, its effect on the
endonucleasesoftheratliverandontopoisomeraseswasstudied(topoisomerasesplay
an important part in the DNA metabolism). In these experiments NSC 631570 inhibited
themetalͲdependantendonucleasesandthetopoisomeraseIaswell(166).


TheEffectontheProteinsInvolvedintotheRemodellingofExtracellularMatrix

AnItalianresearchteamfromtheUniversityofMilanusedRTͲPCR,Westernblotanalysis
and SDSͲzymography to evaluate the effect of NSC 631570 on the expression of genes
and proteins involved into the remodelling of the extracellular matrix. This mechanism
playsanimportantroleinthetumorinvasionofhumanglioblastomacells.Therewasa
significantdoseͲdependantdecreaseoftheproliferationofglioblastomacellsandatrend
to the downͲregulation of secreted protein acidic cysteine rich (SPARC). This study
provided theoretical reasons for the using NSC 631570 in glioblastoma. The authors

37
concluded:NSC631570‘maybeausefultherapeutictoolforbraintumors’(245).Thishas
beenprovedinclinicaluse(see“Braintumors”).

IntheirfurtherworkwithglioblastomacellstheItalianresearchersrevealedNSC631570
to increase the expression of the glial fibrillary acidic protein (GFAP). The connexion 43
expression was not modulated by NSC 631570. These results ‘support the possible
potentialofNSC631570forthetherapyofbraintumors’(250).

ThisItalianteaminvestigatedalsowhetherNSC631570isabletomodulatetheinvitro
expressionofsomeproteinsinvolvedintumorprogressiononthreeclearcelltyperenal
carcinomacelllines(ccRCC).CAKIͲ1,CAKIͲ2andACHNclearcellcarcinoma(ccRCC)cells
weretreatedwiththreedosesofNSC631570(5,10,and20ʅM)orleftuntreated,and
culturedfor48hinduplicate.SPARCproteinlevelswereassayedinWesternblot,MMPͲ2
and MMPͲ9 protein levels and activity were determined by SDSͲzymography in cell
culturesupernatants,andthedistributionofthedifferentcellcyclephaseswascalculated
using FACS analysis. The results suggest that NSC 631570 modulates two major aspects
involved in tumorigenesis of RCC cancer cells, that are extracellular matrix remodelling
andcellproliferation.ThetendencytoMMPͲ2andMMPͲ9downͲregulationinUKͲtreated
cells suggests that UK may decrease RCC cell invasion. Moreover, SPARC protein
downͲregulationinsupernatantspointtoaninhibitionelicitedbyUKalsoonextracellular
matrix remodelling in the tumor microenvironment, possibly rendering the tumor
microenvironment less permissive for tumor invasion. At the same time, SPARC
intracellularproteinlevelsupͲregulationinccRCCcellssuggestthatNSC631570maybe
involved in the inhibition of cell proliferation by cell cycle inhibition, as shown by FACS
analysis. (Pettinari et al, ‘Matrix metalloproteinases activity and SPARC expression are
targetedbyUkrainadministrationinrenalcellcarcinoma’,presentationatthesymposium
‘TargetingCancerInvasionandMetastasis’,Miami,USA,2010).

The effect of NSC 631570 on the modulation of some of the key markers of tumor
progression in pancreatic carcinoma was investigated on three cell lines HPAFͲII, PL45,
andHPAC.ThecelllinesweretreatedwithNSC631570(5,10and20µM)for48h,orleft
untreated. Secreted protein acidic and rich in cysteine (SPARC) mRNA levels were
assessed by realͲtime PCR. Matrix metalloproteinases (MMP)Ͳ2 and Ͳ9 activities was
analyzedbySDSzymography;SPARCproteinlevelsincelllysatesandsupernatantswere
determinedbyWesternblot.Cellcyclewasdeterminedbyflowcytometricanalysis,and
invasion by matrigel invasion assay. NSC 631570 downͲregulated MMPͲ2 and MMPͲ9,
suggestingthatthisagentmaydecreasepancreaticcancercellinvasion,asconfirmedby
thematrigelinvasionassay.SPARCproteindownregulationinsupernatantspointstoan
inhibition NSC 631570 of extracellular matrix remodelling in the tumor
microenvironment. At the same time, SPARC mRNA and cellular protein level upͲ
regulationsuggeststhatNSC631570canaffectcellproliferationbycellcycleinhibition,
showingacellcycleG2/MarrestinUKͲtreatedcells(266).






38