Cisplatin

Cisplatin

CDDP, also referred to as cisplatinum or cisplatin, is a yellow powder and has found widespread use a chemotherapeutic agent. The platinum complex binds to DNA and causes cross-linking, which triggers the programmed cell death (apoptosis). Cisplatin is specifically used as an effective therapeutic agent against ovarian, testicular, uterus, bladder and head and neck cancers (Figure 7.19).

1. Discovery

Rosenberg, a biophysicist working at Michigan State University, discovered the anticancer activity of cisplatin in 1965 serendipitously. Rosenberg devised an experiment to investigate the effect of electric fields on cell division, in which he passed an alternating electric current through two Pt electrodes immersed in a beaker containing Escherichia coli bacteria in a cell growth medium containing ammonium and chloride ions. During the experiment, Rosenberg discovered that the bacteria had grown in size, but not divided as was expected. On carrying out some control experiments, it became soon clear that it was not the electric current that caused this unusual cell growth. Rosenberg realised that a chemical reaction had taken place in the cell medium requiring oxygen, ammonium ions (NH₄⁺) and chloride ions (Cl⁻) in addition to a small amount of platinum, which was dissolved from the surface of the electrodes. A mixture of platinum salts was accidentally synthesised which contained cisplatin (cis-[Pt(II)Cl₂(NH₃)₂]). Rosenberg subsequently showed that only cis-[Pt(II)Cl₂(NH₃)₂] and not trans-[Pt(II)Cl₂(NH₃)₂] could prevent the growth of cancer cells in vitro. Typically, cisplatin kills cancer cells at micromolar doses.

Nevertheless, there is a lot of work necessary in between the discovery of a cytotoxic agent and the licensing of an anticancer drug. At the time Rosenberg discovered the potential of cisplatin, only organic compounds were seen to be appropriate for medicinal use in humans and certainly a heavy metal compound was seen as being too toxic for a therapeutic approach. Rosenberg convinced research institutes such as the National Cancer Institute to carry out several tests and trials. In 1979, he finally managed to file a patent on the use of cisplatin as anticancer agent. The synthesis itself had been reported 100 years ago, and cisplatin certainly was not a novel compound anymore, which could be patented. Bristol-Myers became interested in the compound, and the FDA licensed cisplatin as an anticancer drug. This discovery led to a whole new area of drug discovery, as from this point drug development was not only limited to organic compounds anymore.

2. Mode of action

Cisplatin is a neutral, purely inorganic compound, first synthesised in 1844, containing a platinum(II) centre and two ammonia ligands and two chloride ligands. The ammonia ligands represent the nonleaving groups, whereas the chloride ligands are labile and can be exchanged by nucleophiles (Figure 7.20).

The synthesis of cisplatin starts with K₂[PtCl₄], but it has undergone several improvements since it was published more than 100 years ago. The main problem is the occurrence of impurities and the synthesis of the by-product transplatin. Nowadays, the synthetic routes are mostly based on a method published in the 1970s by Dhara. In the initial step, K₂[PtCl₄] is reacted with KI, and the platinum complex is converted into the tetraiodo analogue. Subsequently, NH₃ is added and cis-[PtI₂(NH₃)₂] is obtained. cis-[PtI₂(NH₃)₂] precipitates from the solution once AgNO₃ is added, and the insoluble AgI can be filtered off. KCl is added to the solution and cisplatin is formed as a yellow solid. The success of the synthesis relies on the strong translabellising effect of the iodo ligands as discussed earlier  (Figure 7.21).

Since the success of using cisplatin as a chemotherapeutic agent, significant research has been undertaken to establish the exact mode of action. Some areas still remain unclear, but it is clear that its anticancer ability mainly stems from its ability to form adducts with DNA. It has been generally accepted that the cytotoxic activity of cisplatin is due to the interaction between the metal complex and the genetic DNA, which is located in the cell nucleus .

Cisplatin is administered intravenously as the neutral complex, and transported via the blood stream to the cancer cell. The blood stream and the extracellular fluids have a high chloride concentration (>100 mM) and therefore, the platinum complex will not be hydrolysed. There is still much debate about the cellular uptake. It is believed that the neutral complex enters the cancer cell by passive and/or active transport. Apart from the passive diffusion, carrier-mediated proteins have been identified, such as the plasma membrane copper transporter, organic cation transporters and others  (Figure 7.22).

The mode of action inside the cell begins with the hydrolysis of the platinum–chloride bonds. This hydrol-ysis is facilitated by the significantly lower chloride concentration inside the cell (4 mM) compared to the high chloride concentration in the blood plasma, which prevents the hydrolysis of cisplatin during the trans-port in the blood stream. Upon entering the cell, it is proposed that cisplatin loses its chloride ligands and forms the mono and diaqua species. The hydrolysed species are good electrophiles and can bind to a variety of nucleophiles in the cell, such as nucleic acid and thiol-containing proteins (Figure 7.23).

The anticancer activity of cisplatin is based on the interaction of the platinum complex with DNA located in the nucleus. Interaction with the mitochondrial DNA is believed to be less important for the antitumour activity of cisplatin. Cisplatin binds to DNA primarily by coordination to the nitrogen (N7) atom of guanine, whereas it also can bind (to a lesser degree) to N7 and N1 of adenine and N3 of cytosine [2, 6] (Figure 7.24).

It was found that the majority of DNA–cisplatin complexes are formed by intrastrand 1,2-(GG) cross-links, which means cisplatin coordinates to two guanine bases within the same DNA strand. This form of DNA-adduct formation makes up around 65% of all DNA–cisplatin complexes. Note that the Arabic numbers refer to two adjacent nucleotides within the DNA sequence and ‘intra’ means that the cross-link occurs within the same DNA strand. Other binding modes include intrastrand 1,2-(AG) cross-links (25%) as well as inter-strand cross-links (between two DNA strands) and mono-functional binding to DNA  (Figure 7.25).

As a result of the formation of DNA–cisplatin adducts, the secondary structure of DNA is affected. In par-ticular, the major intrastrand cross-linking of cisplatin leads to conformational alteration in the DNA. The plat-inum core binds to N7 of guanine bases located in the major groove and, as a result of its square planar geome-try, forces the two bases to tilt towards each other and away from the parallel stacked form of DNA. This leads to a distortion of the helix axis, bending it towards the major groove. In turn, this exposes the minor groove on the opposite site and makes it accessible for other compounds. In essence, the formation of these adducts results in the DNA helix to become kinked and the DNA translation to be interrupted [2, 6, 7] (Figure 7.26).

3. Resistance and cytotoxicity

One of the major problems of chemotherapy with cisplatin is that after repeated use the cancer cells often become resistant to the treatment. Unfortunately, cisplatin has a narrow therapeutic window, which means that the difference between the therapeutically active and the toxic dose is fairly small. Therefore, it is not simply possibly to increase the dose once resistance is observed.

It has been observed that in platinum-resistant cells, less of the metal is bound. This can be due to the cells being more effective blocking entry to the cell, being able to move cisplatin actively out of the cell or, in the case where the agent made its way to its target, that DNA can be repaired and the metal complex is removed.

It has been shown that cancer cells that develop resistance to cisplatin have a higher concentration of the sulfur-containing proteins such as glutathione (GSH) and metallothionein (MT). Both GSH and MT can form very stable complexes with Pt²⁺ via a S—Pt bond, which will reduce the ability of the platinum complex to interact with other targets in the cell. It is actually assumed that most of the platinum drugs bind to sulfur before it reaches the DNA, as this interaction is the kinetically favoured process. There is evidence that some of the Pt–thioester complexes can be broken in the presence of DNA. Nevertheless, the Pt–thiol complexes are very stable and will therefore inactivate the platinum drug . There is also evidence that the Pt(II)–S adducts could be exported out of the cell via a glutathione S-conjugate . It is interesting to note that only about 1% of the administered cisplatin will complex with DNA .

DNA repair mechanisms are important processes within the cells and there are several different ways in which DNA can be repaired. If the DNA has been chemically modified by drugs, UV light or radicals, Nature has established a sophisticated system to check and repair the DNA and ultimately ensure the survival of the organism. 

One system is called nucleotide excision repair (NER) and it uses enzymes to remove the single-stranded DNA segment that has been modified. It also replaces this segment by reading the opposite DNA strand. As previously discussed, the 1,2-intrastrand cross-link represents the major Pt–DNA adduct, where cisplatin coordinates to two neighbouring guanine bases within the same DNA strand. In recent animal experiments, it has been shown that the NER system is indeed important to the removal of platinum from DNA. Mice with a compromised NER system were not able to remove the platinum-coordinated segments from their DNA in the kidney cells researched . There are also other mechanisms of resistance under discussion, which are not further explained in this book.

4. Formulation and administration

Cisplatin is commercially available either as a powder for reconstitution or as a ready-to-use solution con-taining the drug in a saline concentration (NaCl solution) at a pH around 3.5–4.5. Under this condition, the majority of platinum is present as [PtCl₂(NH₃)₂]. Additionally, the low pH avoids the formation of any multinuclear platinum compounds.

Cisplatin is used alone or in combination for the treatment of bladder, lung, cervical, ovarian and testicular cancer. It is administered intravenously and requires pre-hydration of the patient. The dose of cisplatin depends on the body surface of the patient and ranges typically from 20 to 140 mg/m². It can be given as a single-dose injection or as infusion over a period of a couple of hours. Cisplatin is mainly eliminated via urine. The highest concentrations can be found in the liver, prostate and kidneys. Side effects include severe nausea, vomiting, ototoxicity and nephrotoxicity, together with myelosuppression (bone marrow suppression). In particular, nephrotoxicity is the dose-limiting factor, and close monitoring of the renal function is required. The exact mode of damage to the kidneys is unknown, but it accumulates in the kidneys and believed to damage the renal tubular cells [2, 8].

5. Transplatin

Soon the question arose how transplatin (trans-diamminedichloroplatinum (II)) differs from its stereoisomer cisplatin and how this explains the dramatic difference in efficacy. Very early, it was seen that transplatin is not active when tested in animal models, and therefore it was postulated that the cis geometry is crucial for the cytotoxic activity for which two reasons where identified (Figure 7.27).

A close look at the structure of transplatin shows that the two chloride ligands (i.e. the reactive sites) are further apart in transplatin (4.64 Å) compared to cisplatin (3.29 Å). This affects the way the metal centre Pt(II) can cross-link sites on DNA, which means that the 1,2 intrastrand cross-link between the two purine bases is stereochemically hindered for the transplatinum species (transplatin). Studies have shown that mainly the mono-functional 1,3 adducts and inter-strand adducts are formed, which are easily recognised by the NER repair system .

Furthermore, it has been shown that transplatin is kinetically more reactive than cisplatin and therefore more of the compound is inactivated before reaching its target, which contributes to the weaker activity of transplatin. Therefore, current research focusses on the synthesis of novel transplatin compounds in which the hydrolysis rate of the compound can be altered .

Recently, a range of transplatinum compounds were proved to have anticancer activity. The most notable compounds are the trans-Pt(IV) complexes with the general formula trans-Pt(IV)Cl₂X₂LL^′ or the trans-Pt(II) complexes with the general formula trans-Pt(II)Cl₂LL^′. The ligands (L and L^′) are mainly imino, aromatic amine and aliphatic amine ligands, whereas the Pt(IV) complexes contain also hydroxyl groups. Interestingly, some of those compounds are also active on cisplatin-resistant cell lines, thereby opening a new area of research for the treatment of these cancer types [9, 10] (Figure 7.28).