On the Mechanism of Action of the Antitumor Drug cis-Platin (cis-DDP) and its Second Generation Derivatives

The present article attempts to summarise the elements of the mechanism of action of the antitumor drug cis¯-Platin presented the last few years. Highlights on the discovery, of the drug and the development of it’s second generation derivatives are presented, as well as the ways that cis¯-DDP reacts with biomolecules as DNA and proteins and their models e.g. nucleosides, nucleotides. Also the hydrolysis data are presented for cis¯-DDP and its’ inactive congener trans¯-DDP, as well as for the second generation drug carboplatin. Finally, usefull conclusions are given from this work, pointing out the unanswered questions about the action of cis¯-DDP as well as its differences in action, in comparison with trans¯-DDP.


-IntraSCL
Bidentate bindin of cis-DDP with one base of DNA Interaction of cis-and trans-DDP with proteins and formation of DNA-Pt-protein crosslinks.

ELEMENTS OF PHARMAKOKINETICS OF cis-DDP, IPROPLATIN (a) Introduction
The compound dichloro-diamine-platinum (II), of the Cl was known since 1844 in two forms, the empirical formula Pt(NH3) 2 a-Pt(NH3)2C1 2 or Peyrone salt2(ci__s-platin) and the 6-Pt(NH3)212 or Reiset salt (trans-platln). The pioneering work of Werner [I] 6n the structure and bonding of coordination compounds, led to the assignment of a square planar geometry for these complexes (Fig. I). (a) ci__#s-platin or cis-DDP and (b) of trans-effect was made by the Russsian chemist Chernyaev [5].
subsequently studied in depth by Basolo [6], Dra9o [7], Sadler [8] of t rans-p at n ructure of both isomers was elucidated by X-ray diffraction 2]. um (II) is classsified as a "soft" Lewis acid, according to 3] or class b, and generally forms more stable bonds with donor racterized also as "soft" Lewis bas#s (e.g. N,S,P etc), while with ses (e.g. Cl, H20, NO CO SO etc) it forms weaker bonds. s on ligand substitut3 3 4 Ion reactions in square planar complexes led velopment of the kinetic trans-effect by Chatt [4]. The discovery It was etc and it continues to be studied even today.
Around 1965 the first publication appeared by B. Rosenber9 and his collaborators [9] describing the unusual behavior of E.coli bacteria culture, in the presence of platinum electrodes. This was a part of a biophysical study of the growth of bacteria under the influence of the electric current.
The bacteria were forming long filaments, about 300 times longer than the normal size [9]. This filamentous growth meant inhibition of cell division, but not of cell growth.
The platinum electrodes were selected because of the inertness of the metal, while the observed filamentous growth was starting I-2 hrs after the passage of the electric current. It was subsequently found that for this abnormal growth of the bacteria, small amounts of metal in the form of the complex (NH4)2PtCI_ (I-10 ppm) were necessary. The  (ci___s-platin, or cis-DDP) started in 1972, as well as in vitro tesls of various platinum complexes against a broad spectrum of tumors. Thus, from a broad spectrum of 28 tumor types with 50 to I00% cures (phase I clinical trials), 7 types of toxicity effects were discovered, with as more serious ones, nephrotoxicity, nausea and vomiting. Nephrotoxicity was determining the limiting dose of the drug. The best results of cis-DDP were found against testicular and ovarian cancers. The problem of nephrotoxicity on the other hand, was solved by the idea of Cvitkovic and Krakoff at the Sloan Ketterin9 Institute, to use extensive hydration and mannitol prior to drug administration.
Cis-DDP was subsequently used in combinational therapy with vinblastine and bleomycine against testicular cancer with >95; cures.
Analogs of cis-DDP exhibitini antitumor activity were also presented by Rosenber9 in 1978 [14] (Fig. 2). (2)-0, 0' latinum (II), named Paraplatin or Carboplatin, or JM-8 is commercialy widely used (Fig. 3). Figure 3 presents a few analogs of ci__s-DDP with satisfactory antitumor activity that are presently under clinical trials in various phases, except carboplatin, which is already in use. Carboplatin [18].The present paper is attempting to briefly review what is known today on the mechanism of action of cis-DDP and carboplatin, which almost replaced the former and is the only one in use.
156 (b) Structure-Activity Relationship The aquired knowledge on the activity and chemistry of cis-DDP and its inactive congener trans-DDP, even from the early studies and the testing of many analogs of the drug against various tumors, provided the first elements of structure-activity relationship of these complexes , summarized in the folllowin9 rules: (i) T compounds should be neutl and correspond to formulae cis-Pt (am) X or cis,cis,trans-Pt--(am containing no hydro9ens capable to form hydrogen bonds, do not possess antitumor activity, though they can also bind with DNA. The possible roles of the N-H group in the biological activity of the compounds, may either be kinetic (e.9. a possible relationship with the approaching of the macromolecule of DNA) or thermodynamic (e.9. with the additional (de)stabilization after binding with the biological target of DNA) or steric (e.9. hindered rotation, due to the increase of bulkiness of of the nitrogen donor l igand) or reasons related to the transport of the drug within the cell. In any case, it is even today a subject of extensive research [2].
It should be emphasized that the above rules are not universal and that some Pt(II) compounds with increased cytotoxicity or even antitumor activity do not obey them [28]. A few examples are liven below: (i) Complexes with two diamine units bridged with a carbonate bridge of NH )}] [29][30][31]   und that DNA was the target molecule of ci__s-DDP within the vo, because the drug had the following biological effectsn of a filamentous growth in bacteria, (i_i) the production (iii) carcinogenicity, (iv) inactivation of viruses and ion of bacteriophage viruses that contain and transport Cis-DDP had also the following biochemical effects supporting the above conclusion-(i) selective inhibition of the synthesis of DNA and not of RNA or proteins, (ii) inactivation of the substrate of the enzyme DNA-polymerase, (iii) selective binding with DNA, (i_v) other analogous complexes are bound in a similar manner to cis-DDP with DNA and finally, (v) the relationship between the antitumor activity and inhibition of the growth of bacteria [42].
The fact that the nuclear DNA was the primary target of cis-DDP within the cell, led to extensive research of the study of the interaction of the drug and other platinum salts with DNA and its constituents in vitro with the aim to discover the binding sites of the metal, its general reactivity and the possible modifications of DNA caused by the binding, leading to death of the cell.
In 1971, the first publication on the interaction of cis-DDP with DNA, studied with UV and CD spectroscopies appeared [43]. The hypochromicity shown in the UV spectra of the double helix of DNA, indicates perturbation on the structure of the macromolecule upon platinum binding, i.e., the disappearance of the stacking interactions between the bases (increase of ). This was interpreted as rather due to a covalent bonding of Pt(II) with the bases of DNA. At almost the same time the ir spectrum of the complex ci__s-Pt(ado)Cl (ado=adenosine) was studied in the solid state, with Pt(II) bound at t.: 7 atom of the base [44].
Various techniques were employed in subsequent studies of interaction of platinum salts with nucleosides and nucleotides (DNA constituents), leading to the conclusions that the metal could mainly bind at the N7 and NI sites of purines and the N3 site of pyrimidines [45][46][47][48][49][50][51][52][53]. Later similar studies of nucleobases showed additional binding sites the N3 and 06 of purines and the exocyclic amino group (-NH) of pyrimidines [54, 55] (Fig. 4). Pt and N-NMR [60, 62, 63] have been used in many cases to identify the species formed, while many hydroxo-complexes were isolated in the solid state and their structure was solved by X-ray diffraction [17, 64-66].  The within take pl atoms o than Pt place t Its is not a 9ood leaving group as compared to H O, the concentration of the trans-mono-hydro-complex available is considSrably reduced, consequently, its reaction with DNA is also reduced [56].
blood plasma has pH=7.4 with chloride concentration of 100mM, while the cell it is only 4 mM. Consequently, the hydrolysis reactions ace in the cell plasma, by the hydrated species with the proper f the biomolecules.
It is noted that Pt(II) is hydrolyzed faster (IV) and that the l igand substitution reactions in the former take hrough a pentacoordinated intermediate [57].
hould also be noted that the species distribution of Figs. 5,6 is not strictly followed in vivo, since it is possible for the complexes to react with the various biomolecules, e.g. proteins of blood serum, in sulfur containing sites (glutathione, methionine etc) [67-70], prior to reaching thermodynamic equilibrium.
The hydroxo bridged species, that may also be formed in regions with high platinum concentration [71], are inert and believed to be related to the neurotoxicity of the drug. The release of ammonia from cis-DDP when the drug reacts with sulfur donors, like methionine [72], rises the question whether this release contributes to its cytotoxicity [72, 73].

BINDING MODES OF METAL IONS WITH DNA (a) Introduction (_Description of DNA structures)
The secondary structure of the B-form of DNA is shown in Fig. 7. Part of its primary structure with the possible sites on the bases for coordination with platinum is also given in Fig. 8. (.ii) Base pairs per turn-It is a measure of the magnitude of the helix and represents the total number of bases after a complete turn of the helix, i.e. after arriving again to the origin where it was started from (For B-DNA we have 10 pairs per turn, i.e., per 10 X 340 = 3400 pm).
(iii) Helical pitch Distance run from the origin of the helix, required for a complete turn (in B-DNA is 3400 pm or 34 ).
(iv) Base tilt Angle of the plane of base pair with the perpendicular to 00 the axis of the helix plane at the same point (In B-DNA it is i.e., every base pair determines a plane perpendicular to the helix axis or otherwise the planes determined by the bases are perpendicular to the axis).
(v) Base displacement Distance of the centre of the base pairs from the helix axis (0 pm in B-DNA).
The B-form is the most common in solution. There are also the right hand side A-helix, that has a total number of 11 base pairs per turn, base tilt of 20 and the left hand side, Z-form (zig-zag tpe), which is more scarce. On a B-helix, the axis of every base pair (i.e., the straight line connectin the centre of the bases) is turned in relation to the previous pair by 36 (e.9. the angle of the two axes is 36). In this way a major groove and a minor groove are finally formed in the macromolecule.

The 161
On the Mechanism of Action of the Antitumor Drug cis-Platin (cL-DDP) and Its Second Generation Derivative, o first (major groove) is 24 A wide and it is considered as the primary rec_ognition site of the proteins, while the second (minor groove) is onl 10 wide with smaller depth and interacting onlM with small proteins, In the B-helix the minimum distance for two bases belonging to two different helixes is 7 Intercalators are planar molecules, mainl organic polyaromatic systems, havin the abilit to intercalate between two successive base pairs kept near, b9 stacking interactions. A DNA-intercalator "complex" is thus formed. This possiblit of course exists on Iw for a ds DNA and not for ss DNA. The effect of intercalation in the geometr of the molecule is the lengthening of the helix [70], so that the distance between the base pais in the two sites of the intercalato is increased to 7 [42]. Also, the complex [Co(NH3)6] or the Mg(H_O) 6__ are known to stabilize the Z-form of DNA through a B->Z transition [90].
Greater stabilization is achieved with the first complex, because (a) it has a larger charge, (b)  More particularly, cis-DDP can bind with ways ( Fig. 11).  (b) transplatin Various ways of (_a_a) DNA binding of cis-DDP or/and proteins and (b) differences in the conformation of the macromolecule from the formation of 1,2or" 1,3-TntraSCL by cis-DDP and 1,3-or 1,4-IntraSCL by tr'ans-DDP.

cisplatin
The percentage formation of each type of bonding were quantitatively calculated [98,99,100]. Thus the percentage of the intrastrand crosslink betweeen two successive bases of guanine of the same strand of DNA (1,2-IntraSCL) formed by cis-DDP on the d(GpG) part of the macromolecule, varies from 50-60%, whereas IntraSCL between successive guanine and adenine bases (d(ApG)type) by 20-30 percentage. About 10% of cis-DDP forms 1,3-InstraSOL with two guanine bases separated by a third base of the type d(GpXpG). More scarce are the InterSCL between guanine bases belonging to different strands of DNA (<I;) and the DNA-protein crosslinks (<1%).
More types of binding are also taking place in small percentages, but they have not yet been identified [98,99]. The I/2 monohydrated species produced, react fast mainly with the N7 of guanine of DNA, which is not only nucleophilic but also easy to be reached by the salt, in the direction of the major groove in B-DNA.
Experimental evidence for the formation of intraSCL in DNA between two N7 atoms of neighboring G molecules, also referred as a 1,2-1ntraSCL, are provided by the following series of experiments: (_i) A large increase is observed in the value of the flotation density for the polynucleotide, poly(dG)poly(dC) that was treated with cis-DDP, compared to the corresponding increase of the platinated poly(dG.dG) and the platinated monomers.
Both poly(dG.dC) and poly(dG The fact that only poly(dG).poly(dC) contains adjacent guanine bases, shows the importance of the ,2-IntraSCL (large increase upon platination in the flotation density value).
Trans-DDP increases the flotation density of DNA proportionally to its G-content, but it does not increase it so much for poly(dG).poly(dC), in contrast to cis-DDP, i.e., it is not showing any selectivity in the formation of IntraSCL, in neighboring bases [104]. (j_i) The breaking of platinated DNA with enzymes possessing selectivity, e.g., restriction endonucleases. This selective breaking is inhibited by cis-DDP when the sites cut by the enzyme, have nearby the sequence, oligo (dG).oligo(dC) [105][106][107].
Also in experiments with exonucleases and with the use of techniques for sequence analysis of DNA, it is seen that the breaking of the platinated DNA is inhibited in oli9o(dG) sites, most probably due to the formation of IntraSCL with cis-DDP [106][107][108][109].
(iii) NMR spectroscopic techniques on the interactions of di-and oli9onucleotides with cis-and trans-DDP show the formation of IntraSCL. (i_zv) Binding of the two isomers cis-and trans-DDP with DNA followed by hydrolysis (mainly enzymatic or other) and identificatio9 of the obtained products with chromatographic HPLO and FPLO analysis and HNMR, showed for 1 cis-DDP the existence of only 1,2-IntraSCL of the GG and AG but not of the 6A (5'----3') type [99,110,111].
Thus, HNMR and chromatographic methods of the reaction products of cis-DDP with various homoand heterodinucleotides showed that there is a kinetic selectivity in the reaction with d(GpG), while the reaction with d(ApA) was very slow [112].
In the case of the reaction of ci__s-DDP with d(GpC), a 1-2 complex of the metal with the ligand is formed, bound only through N of G. Cytosine is not taking part in the complexation, under the conditions of the reaction, showin the lack of selectivity for a 1,2-Intra$CL with d(GpC).
The similar t l2 of the monodentate complexes with A of both isomers, show that the bioSo91ca] nactivity of trans-DDP cannot be due o the rate of the formation of a bidentate complex [116]. It can however be related to the reaction rate of su]fydry] (SH) 9roups with the monodenta]]y bound to DA trans-DOP, which is faster in comparison ,ith the ones of cis-DDP, as it is seen ith reactions of both ,ith 9]utathione (6SH) [118].  [119]. structure determinations have also been carried out on some of ion products of cis-and trans-DDP with ol ilonucleotides. Very was the elucidation of the crystal structure of the reaction f cis-DDP with d(pGpG) forming a 1,2-IntraSCL [120,121] (Fig. 13).
Other crystallographi studies include the structures of cis-and trans-DDP with t-RNA pne from brewer yeast with 6 and 3 resolution, respectively [124,125]. Cis-DDP formed 1,2-IntraSCL of the type d(GpG) and d(ApG) [124], while trans-DDP [125] is monodentally bound at G(34) [125]. Also in the structure of [(dien)Pt[d(ApGpA)-NT(2)], complexation takes place only through N7 of G(2) (Fig. 14).  [126] on the ci__s-DDP adduct of ype d(TCTCGGTCTC).d(GAGACCGAGA) containing 7 of G(5) and G(6) showed that two bending lix axis in the major groove exist, ngles of the two complexed bases are y [127]. The hydrogen bondin between two complexed guanine bases with their complementary cytosines is modified. The sugar conformation is changed to C3'-endo from C2'-endo with then the also for the platinated 9uanosines and a hydrogen bonding exist between the ammonia molecules of cis-DDP and the 5'-phosphate group. Of the two bending models for the structure of B-DNA in the presence of cis-DDP, the one o corresponding to 63.2 is more stable by 19.3 kcal/mol under high ionic strength conditions (i.e., with maximum neutralization of charge of DNA), called "model of high salt concentration", while in lower ionic strength the 58.4 model is more stable, called "model of low salt concentration" [126]. Similar molecular mechanics calculations for the binding of cis-DDP with nucleotides of the type XpG and GpX, (X-base), show selectivity for platination when X=G [128]. The [133] show that this latter type of binding should be about 5 times more toxic than the former one. This indicates that we have to be cautious in determining the more cytotoxic IntraSCL of the drug. During the replication of platinated DNA, the 5-position of the sugar presents the larger distortion, mainly due to the structural change to the C3'-endo conformation of the latter.
As a consequence mutation may occur at this position (replacement of C by A) and not at the 3' where the smaller (maintenance of the C2'-endo configuration) [132] distortion is ( Fig. 15). A comparison of the effects caused by platination (cis-DDP) and methylation at the N7 position of dGuo and dGpG, shows that the former stabilizes more than the latter the N-glycosidic bonding towards acid 170 hydrolysis and does not cause the opening of the imidazole ring [134]. This means that the N7 platinated 9uanines are slower hydrolizin9 the N-91ycosidic bonding than the methylated ones, e.9. slowly apopurinate from DNA. Also the difference from a nucleoside (dGuo) and a dinucleotide (GpG) upon platination consists to the easier breaking of the N-glycosidic bonding in the former and of the Pt-N7 bonding in the latter [134].
It was finally proposed that for the formation of the 1,2-IntraSCL at the d(GpG) section of DNA, the cis-DDP is initially accidentally bound with one guanine base monodentally through N7 in a reversible fashion. In this way, ci__s-DDP may "march" on the double helix until it is able to form a permanent 1,2-IntraSCL when it "finds" two adjacent guanine bases [135].
(c) In.terstrand Crossl inks (InterSCL) Usual ly the percentage of InterSCL is less than I; of the amount of cis-DDP bound with DNA, though it seems to increase with time up to 3% [136] or even up to 5% according to others [137]. Both isomers cis-and trans-DDP form such bonds in comparable amounts [106]. It should be noted that the covalent binding of cis-DDP with the DNA bases creates local unwinding of the double helix, resulting to regions of single stranded structures within the macromolecule and to a final shorternin9 of the active length of it [97].
In DNA rich in G,C, it was proposed [138] that the initial formation of a N706 chelate with cis-DDP of guanine has as a result the breaking of the hydrogen bondings between the two bases G,C in opposite strands. This was followed by the formation of interstrand crosslinks between the deprotonated NI atom of guanine and the N3 of cytosine [138]. This was however, never confirmed. The formation of InterSCL between two guanine bases of opposite strands, bound to Pt(II) through their N7 atoms create an important distortion of the secondary structure of DNA, as molecular three dimensional models show.
More particularly the two helixes are turned in opposite directions approaching each other and the distance between the 91ycosidic bonds is reduced to 10 from 12 X.
When cis-DDP reacts with the oligonucleotide d(CTTCTCCTTGCCTCTCCTTCTTC) and after isolation and indentification of the products with gel electrophoresis and molecular models, it was found that the sequence d(GC/CG) which was platinated was distorted and a bending of 55 was observed in the direction of the major groove. The average angle of turn of the bases is kept constant however. This occurs, because for the 0 formation of the InterSCL the two guanine bases should approach to 3.4 A (bite distance in cis-DDP) from the 7 X, which is their normal distance in B-DNA [140]. The InterSCL may not play a very important role in the cytotoxicity of cis-and trans-DDP [141] or of ci__s-and trans-diamino-tetrachloro-platinum (IV) [142], since it is only formed in a very small percentage, though more recent results show the opposite [143,144]. H NMR studies on the interaction of the trinucleotide d(GpCpG) trans-DDP showed complete unstacking of the bases, while the sugar of changes conformation from C2'-endo to C3'-endo [145]. This is similar the change caused to the sugar at the 5'-position, in the 1,2-IntraSCL ci__s-DDP with d(Gp6), but differs from the 1,3-IntraSCL of cis-DDP with d(GpCpG) [146] favoring the C2'-endo conformation. This may be related the lack of antitumor properties of trans-DDP.  (7) can be detected. When the double stranded 12-nucleotide d(CCTCGAGTGTCTCC).d(GGAGACTCGAGG) is used instead, it is seen from the molecular models of the free and platinated nucleotide (Fig. 16) that because of the 1,3-1ntraSCL, the A(6) at the middle is not anymore stacked with the complexed guanine bases Ca) surrounding it and its sugar takes the C3'-endo conformation [14].
The difference from cis-DDP [127] is due to the fact that this causes a considerable bending of the double helix in the main groove of B-DNA, by 400-70 because of the 1,2-IntraSCL, while the trans-DDP bending angle is only 185, because of the 1,3-IntraSCL.
The rate constant of the formation of t_h 1_,-Intra$CL, with the nucleotide of trans-DDP was, k:(12.5+0.4) _ 10_ISeC while with DNA had comparable value of k=(9.6+0.4) X 10 sec [115], as measured with Pt NMR spectroscopy.
-5 -1 Atomic absorption spectroscopy 9ave the value k=(5.4+_0.4) XlO sec [115], for the rate constant of the reaction of DNA with trans-DDP [148], while it is reminded that the rate constant for the replaemen of the first chlorine by a water molecule (Fig. 6) was k.'=1.9 X 10 sec This l indicates that trans-DDP binds to DNA in two pseudofirst order steps, the first being the chlorine replacement and the formation of a monodentate complex with the N7 of guanine and the second, which is faster, the formation of the 17-membered chelate through the N7 atoms of e(5) and of the nucleotide [148].
The rate of the total reaction seems to be determined from the replacement of chlorine by water.
The formation of a bidentate complex by the two isomers was considered until today as non reversible. the reaction o.f the 12-nucleot pH and after enzyric hydroly HPLC and H and Pt NMR spe G{6) and G(8) was formed in 1,4-IntraSCL between the N3 of solutions [149]. The equilibr was found to be 3, with prefe half life tim. of the 1,3-Int 62C, with AH-91_+2 kj/mol and In conclusion, the 1,3-Int t rans-DDP does not seem to be Recently however, it was found that during ide 5'-d(TCTACGCGTTCT) with trans-DDP at low sis of the products and characterization with ctroscopy, a 1,3-!ntraSOL between the N7 of itially, followed by a rearrangement of a C(5) and the N7 of G(8), in neutral aqueous ium constant of this rearrangement reaction The at fence to the formation of 1,3-IntraSOL. raSL was 1.29 hrs at 30C and 3.6 hrs AS =-58+8 J/mol K [149]. raSCL type bonding formed by both cisresponsible for the antitumor action of former, although it partially inhibits the DNA synthesis. Also, formation of the IntraSCL should not be considered as non reversible, in the case of the importance of the investigatioon [149]. After 12 more days new products with u = 8.50 and 5 u = 8.54 ppm are observed, which may be due eii:her to NTlhelates or NI','( bridged complexes [158].
Cytosine, a pyrimidine base, can also form a four membered chelate ring between the N3 atom of the ring and the exocyclic amino group at 4 [159].
Macrocycle chelates may also be formed bween the N7 of 9uo and the phosphate group and cis-DDP in DNA as P NMR studies showed.
The phosphorous signal is shifted downfield by 1.4 ppm and this is not observed with trans-DDP 160].
This result was also more recently interpreted as due to a particular conformation of the oligonucleotides called hairpin-like [161,162].
Indications for the formation of such bonds have also been 91ven for the reaction of cis-DDP with GMP, with fast atom bombardement mass spectroscopic techniques, on the products of the reaction of 1:1 stoichiometry [163].
In conclusion, the N706 chelate of guanine or any other oxopurine with cis-DDP has never been found yet in any crystal structure solved with X-rays, that unequivocally would prove its existence.
Therefore the formation of such a chelate in vivo seems unlikely [164].
.(f) Interactions of cis-and trans-DDP with p.roteins and formation of DNA -Pt-protein crosslinks Cis-DDP reacts with plasma proteins and about 50% of the drug is bound there one hour after administration [24]. The main binding site of the drug is the sulfur of 91utathione [165][166][167] or the sulfydryl group of cysteine. This binding was correlated with the higher nephrotoxicity of the drug compared to Iproplatin and Paraplatin [167].
Due to the high trans-influence of PIII), ammonia liberation is observed in such reactions in the plasma, by N NMR spectroscopy [72].
Trans-DDP is bound to a larger extent than cis-DDP by histone proteins [168], while cis-DDP reacts faster with non-histone proteins [169].
More specifically, it was proposed that cis-DDP is forming selectively crosslinks between the low molecular weight and high mobility group proteins HMG1 and HMG2 and DNA and that the bonding with the latter takes place on the main groove, while the proteins retain an e-helix structure [175].
Cross]ink bonds are also foraed by both isomers wit.h non-histone prot.eins of HeLa ce]]s, t.hough l:rans-DDP t.o a larger exl;ent,. Both isomers bind 1;o a larger proport.ion t, han in hist,one prot.eins due t.o the 9reat,er methionine content of the latter [176,177]. Such crosslinks have been correlated with the antitumor or cytooxic action of he drug [178][179][180]. Since heir percentage is small however, this seems unlikely today [95].
Also, the DNA-Pt,-proein cross]inks for cis-DDP are more resisan o repairing enzymes than those for trans-DDP [181].
Furthermore, the formation of more DNA-Pt-g]uahione cross]inks of cis-DDP than of trans-DDP, was explained wih the lower toxicity of the drug, since in his way it forms a smaller number of 1,3-[ntraSCL with DNA [182]. Due to the easier formation of DNA-P-protein cross]inks by trans-DDP, the latter was used o map regions of t-RNA in he vicinit,aith,, a protein.
For example, such a cross]ink is formed between the t-RNA of the yeast of Saccharomyces cereviase (G) and of the protein aminoacy]o-t-RNA-nva]--synthetase [183]. The sites of the cross]inks of trans-DDP are the ones of t-RNA va that strongly interac with the protein and in this way they are determined [183]. Proteins play an important role in he formation ot' a "complex" with DNA, once the IntraSCL ot 1,2-type wih cis-DDP are formed. For example, it is known that the tripeptide LysTrpLys form a stacking complex only with DNA treated with cis-DDP but not with trans-DDP.
This was attributed to the formation of certain parts with primary structure in the macromolecule [184].
Extensive work was done afterwards for determination of the structure of these proteins. :r was found [187][188][189][190]  The proteins have a spheric structure with a positively charged nucleus able to "bind" with DNA, through its negative charges, with electrostatic interactions [187]. They have the ability (i) to bind strongly with the primary rather than the secondary structure of DNA [191,192], (ii) to destabilize or to unwind the DNA double helix [193], (iii) to promote superhelix formation of plasmids and (.i_yv) to replace histone proteins when they "bind" with DNA [187].
On the Mechanism of Action of the Antitumor Drug cis-Platin (cis-DDP) and Its Second Generation Derivatives 189] that they are forming two -helixes selectively also recognize DNA of crossed the superhelix formation of natural DNA It is believed that the 1,2-1ntraSCL of cis-DDP are protected by these proteins and in this way are not "recognized" by cell enzymes leading to the final death of the cell. This theory is under extensive investigation [190]. To this behavior of the proteins, the negative charge of their terminal carboxylate groups, at physiological pH, may play an important role in the ability to form hydrogen bonds [187][188][189].
The 26 first aminoacids of two such proteins have been coded, with MW 25.6 and 28 kiloDaltons and correspond to HM-I and HMG-2, respectively [194].

ELEMENTS OF PHARMOKOKINETICS OF cis-DDP PARAPLATIN AND IPROPLATIN.
Cis-DDP and carboplatin are administered intravenously in maximum doses of I00 and 400 mg/m2 respectively, every 4 weeks.
The first is administered in a freshly prepared solution in distilled water with mannitol in 1-I stoichiometry.
Paraplatin is diluted with a 5% dextrose solution in water or with a 0.9 NaCl or with distilled water. After 3 hrs from the administration of cis-DDP, about 90% of the drug is bound with the plasma proteins, while in hr, 50% of it is bound [195]. Paraplatin on the other hand is bound 50% only after about 6 hrs (Table I). In conclusion, in the case of cis-DDP a prohydration of the patient is needed and the intravenous administration is slow (solubility I mg/mol in 0.9% NaCl solution), requirino often admission of the patient in hospital and consequently, increasing the cost. Toxicity, nausea and vomiting tendency appear and are treated with the administration of high doses of antiemetic agents. On the contrary, with Paraplatin there is no need of prohydration. Owed to higher solubility, the administration is fast and the patient is not staying in hospital. Most important, its toxicity is smaller, with myelosuppression the dose limiting toxicity, while nausea and vomiting are treated with lower doses of antiemetics. Therefore paraplatin or carboplatin shows comparative advantages than cis-DDP having lower toxicity, thus allowing increase in dose up to 400 m9r/m2 and lower cost of therapy. Also, its aqueous solutions (0.9 NaCl) 176 are stable for a long time and they should not be freshly prepared before use, like cis-DDP.
The mechanisms, of action of cis-DDP and carboplatin are similar, i.e., carboplatin like cis-DDP react with DNA and form InterSCL and IntraSCL, as well as lntraSCL of DNA-Pt-protein type [196]. In aqueous solutions, the 1,1-cyclobutane dicarboxylic anion is liberated in two steps, with breaking first of one Pt-O bonding and simoultaneous nucleoph]lic addition of water 41 c I owe (or of other ligands, in vivo) wit+k1:8.0X10-seca 2 1 by its complete release with k^=k[ and k=1.BIX10dm mol s (acid catalyzed). The inverse reactions (formation of the chelate) s not taking place in the presence of acid [197].
The rate constant 7fo the overall hydrolysis of carboplatin was 0 estimated to be 7.2XI0 s in phosphate buffer solution of pH-7 at 37 . As exposed, it seems that the reaction of carboplatin with DNA proceeds in two steps, i.e., formation of a monodentate complex first and a bidentate afterwards. Based on the hydrolysis constants, the quantity of carboplatin required to achieve the same percentage of binding with DNA, as cis-DDP, was calculated to b aout 100 times larger than the latter (under similar conditions, k-8X10-s-), although in vivo a 20-40 fold excess seems to be enoulh [198].
In summary carboplatin, (i) is about 45 times less toxic than cis-DDP, (i__i) the IntraSCL that it forms with DNA appear 12 hours later than those of cis-DDP and (iii) the IntraSCL DNA-proteins that it forms show their maximum quantity 6 hours later than the corresponding ones with cis-DDP [ ].
19"H NMR studies show that cyclobutane is rotating fast around the carboxylate groups, bonded to Pt(11), in carboplatin [200]. lt6wa also shown [201] that carboplatin reacts faster with 5'-GMP (k=4.1X10-s-) than the phosphates (k=4,alO-Ts-1) and chloride (k=1,210 -6 -1 s ) or water (k<5.9 X lO-Us ). Once the 1-1 complex of the metal ligand is formed, the formation of the 2 complex through N7 is fast (k=1.3X10 -5 "1 Iproplatin (CHIP) on the other hand containing Pt(IV), acts as a prodrug, to produce the corresponding complex with Pt(11), which then reacts with DNA. This is seen from the fact that for its reaction with DNA in vitro, the addition of a reducing agent like Fe(ClO4)^.6H20 or ascorbic acid is required [202]. On the Mechanism of Action of the Antitumor Drug cis-Platin (eis-DDP) and Its Second Generation Derivatives Despite the great deal of work performed on the subject, there is no definitive conclusion concerning the mechanism of action of cis-DDP, since there are still unanswered questions e.g., the non recognition of the 1,2-1ntraSCL by cell repairing mechanisms, while they are related with DNA replication. It can however be argued that the 1,2-IntraSCL may consitute the main damage caused by cis-DDP to DNA, thereby leading to antitumor action.
The ability of cis-DDP to form 1,2-IntraSCL with d(ApG) and d(GpG) and the inability of trans-DDP to act similarly and form only 1,3-IntFaSCL with d(GpXpG) as well as InterSCL, may also be the main difference of the two isomers, leading to antitumor action for the former and to toxicity only for the latter. Cis-DDP causes a greater distortion to the secondary structure of DNA, while trans-DDP causes a greater local distortion, i.e., near the platination site.
The conformation of the sugar at 5' may also play an important role in the mutagenic and toxic dose of the drug during the formation of the I, 2-I r'Ft raSCL.