ANTICANCER RESEARCH 25: 3031-3040, (2005) ______

Systemic Lipoplatin Infusion Results in Preferential Tumor Uptake in Human Studies

T ENI BOULI K AS1, GEO RGE P. STAT HOPO U LOS 2, N IKOL A OS VO LAKA K IS1 and MARIA VOUG I OUKA 1 ,

1Regulon, Inc. 715 Nor th Shoreline Blvd., Mountain View, C alifornia 94043, U.S.A. and Regulon AE, Grigoriou Afxentiou7, Athehs 174 55; 1Errik os Dunant Hos pital, Mes ogeion 107, Athens 15128, Gr eece ;

Abstract. LipoplatinTM, a liposomal formulation of , Key words: Lipoplatin, cisplatin, tumor targeting, gastric cancer, colon was developed with almost negligible nephrotoxicity, ototoxic ity , cancer, hepatocellular cancer. and ne urotoxicity as demonstrated in prec linical and Phase I human studies. A polyethy le ne-gly col c oating of the liposome w ith a m odel where Lipoplatin damage s more tumor c ompare d to nanoparticles is s upposed to r e sult in tumor accum ulation of the normal c ells. In c onclusion, Lipoplatin has the ability to drug by extravas ation through the altere d tumor vasculatur e. We prefe r entially conce ntr ate in malignant tissue both of primar y and e xplored the hypothe s is that intrav e nous infusion of Lipoplatin m etastatic origin following intravenous infusion to patients. In r esults in tumor targeting in four independent patient c ases (one this respect, Lipoplatin emerges as a very promising drug in the w ith hepatocellular adenoc ar cinom a, two w ith gastr ic cancer, arsenal of chemotherapeutics. and one with colon c ancer ) w ho under went Lipoplatin infusion followed by a pr esche dule d s urger y ~ 20h later. D irec t m easurem ent of platinum le vels in s pecim e ns fr om the exc ised Cis-dichlorodiammineplatinum (II) (Cisplatin) continues to play tumor s and normal tissues showe d that total platinum lev els w ere a central role in , for the treatment of epithelial on the aver age 10-50 times higher in m alignant tiss ue c ompare d malignancies, for over 20 years, since its serendipitous to the adjacent normal tissue s pecim ens; most effec tive targeting discovery in 1965 (1) and its identification in 1969 (2,3, w as observ e d in colon c ancer w ith an accumulation up to 200- reviewed in 4,5). The antitumor properties of cisplatin are fold higher in c olon tumors compare d to normal c olon tissue. Of attributed to the kinetics of its chloride ligand displacement the s e veral s urgic al specime ns , gastric tumor s displaye d the reactions leading to DNA crosslinking activities; indeed, once highes t lev els of total platinum suggesting Lipoplatin as a inside the cell, the lower chloride ion content of the cytoplasm c andidate anticanc er agent for gastric tumors; gastric tumor allows the two chloride groups of cisplatin to exchange with s pecim ens had up to 260 m icrogr am s platinum /g tiss ue that w as water yielding the diaquo (hydroxo) species. Although cisplatin higher than any tissue lev el in animals treate d at much higher reacts directly with sulfur groups (such as glutathione) the doses . Fat tissue display e d a high accum ulation of total platinum reaction of cisplatin with DNA depends on its prior hydrolysis to in surgical spe c imens in three differe nt patients c orre lating to the hydroxo complexes that are much more reactive with nitrogen lipid capsule of c isplatin in its Lipoplatin formulation. It was also and oxygen donor groups on proteins and DNA. The diaquo inferr ed that normal tissue had m ore platinum trapped in the species of cisplatin was found in early studies to react with tissue but not r eacted with macromolec ule s where as tumor tiss ue pyrimidines and substituted pyrimidines and to achieve displaye d platinum that re acte d with cellular macromole c ules; the spectacular cures of ascites Sarcoma 180 tumors in Swiss mice data wer e consis te nt (6). Intensive work toward improvement of cisplatin, and with hundreds of platinum drugs tested resulted in the introduction of carboplatin and of oxaliplatin used only for a very narrow ______spectrum of cancers. Cisplatin, carboplatin, oxaliplatin and most other platinum Correspondence to: Dr Teni Boulikas, Ph.D., Regulon, A.E., 7 compounds induce similar types of damage to DNA and their Grigoriou Afxentiou, Alimos, Athens 17455, Greece; Tel: +30-210- tumor killing properties largely depend on their ability to induce 9858454; Tel/Fax: +30-210-9858453, e-mail: [email protected] apoptosis; this is mediated by activation of signal transduction leading to the death receptor mechanisms as well as

3031 ANTICANCER RESEARCH 25: 3031-3040, (2005) ______mitochondrial pathways. Cisplatin has the talent to elicit patients (25). The mechanism of ototoxicity involves loss of crosslinks in DNA confronting the DNA repair machinery. inner and outer hair cells of the basal turns of the cochlear, loss Furthermore, cisplatin alters signal transduction and apoptotic of spinal ganglion cells and degeneration of the stria vascularis pathways crosstalking to other signaling pathways such as those (26,27) caused by induction of apoptosis in these cell types leading to upregulation of transcription factor and (reviewed in 8). nuclear/cellular enzymes (reviewed in 7-9). The unique anticancer properties of cisplatin have prompted Platinum drugs elicit a distinct pattern of side effects numerous attempts to either improve its therapeutic efficacy or depending on their reaction with blood components, tissue to reduce its side effects without compromising therapeutic biodistribution, half-life in plasma, ability to cross the cell efficacy. Modifications have focused either in formulations of membrane in tissue targets, and molecular mechanisms cisplatin aimed at enhancing its tumor targeting to minimize including, reactivity to various types of macromolecules damage to nonmalignant tissue and to lower the side effects or to especially DNA, type of DNA reaction products, extend of chemical modifications on the molecule to derive molecules of damage to normal compared to malignant tissue, rate of repair of better qualities. DNA damage by particular cell types and a number of other LipoplatinTM, is a novel liposomal formulation of cisplatin parameters. The clinical use of cisplatin is impeded by a cute formulated into liposomes composed of dipalmitoyl toxicities , s uch a s nausea a nd vomiting (GI tract toxicity), and phosphatidyl glycerol (DPPG), soy phosphatidyl choline (SPC- cumulative dose-dependent chronic side effects of 3), cholesterol and methoxy-polyethylene glycol–distearoyl nephrotoxicity, ototoxicity, a nd ne urotoxicity as a result of phosphatidylethanolamine (mMPEG2000-DSPE). In a previous damage of cells in these tissues (reviewed in 7,10). Since the work Lipoplatin was shown to be less toxic than cisplatin in reduction of the renal side effects with hydration and of the animals and to cause reduction of tumors after intraperitoneal or gastrointestinal side effects with antiemetics, neurotoxicity is the intravenous injection to human breast MCF-7 or prostate LNCaP most important adverse effect associated with cisplatin xenografts (28); histological examination of the treated tumors chemotherapy occurring in 47% among 292 from mouse xenografts was consistent with apoptosis in the patients treated with cisplatin combinations at conventional tumor in a mechanism similar to that of cisplatin. Mice and rats doses in randomized controlled studies compared to 25% of injected with cisplatin developed renal insufficiency with clear patients treated with the non-cisplatin-containing regimens (11). evidence for tubular damage, but those injected with the same The symptoms include numbness, tingling, paresthesiae in the dose of Lipoplatin were free of kidney injury (29). In a Phase I extremeties, difficulty in walking, decreased vibration sense in study involving 27 patients Lipoplatin dose escalation was the toes, deep tendon reflexes, loss of the ankle jerks, difficulty administered as second or third line chemotherapy; Lipoplatin with manual dexterity, difficulty with ambulation from a deficit showed no nephrotoxicity and it lacked the other serious side in proprioception and gait disturbances; less frequent symptoms effects of cisplatin up to a dose of 125 mg/m2 every 14 days; the from cisplatin neurotoxicity include retrobulbar neuritis, MTD was above 350 mg/m2 as a single infusion (30). encephalitic symptoms, autonomic neuropathy, cerebral Because Lipoplatin nanoparticles bear a PEG-coating it can herniation, seizures, cortical blindness, ophthalmologic effects, be inferred that intravenous infusion of this drug shall result in and vertigo (12-15). Unfortunately, neuropathy is long-term with its preferential distribution at tumor sites via extravasation significant worsening of the symptoms in the first 4 months (16) through the leaky tumor vasculature in analogy to PEGylated that may persist for over 52 months after stopping cisplatin DOXIL/Caelyx liposomal doxorubicin (31). The objective of the treatment (17). Higher platinum concentrations in tissues from present study was to investigate the possibility of platinum the peripheral nervous system (peripheral nerves and dorsal root accumulation in solid human tumors after Lipoplatin infusion in ganglia) compared to tissues from the central nervous system cancer patients. Single doses of 100 mg/m 2 Lipoplatin were used (brain, spinal cord) seem to correlate with clinical symptoms of that are known not to cause any adverse reactions (30). peripheral neuropathy (18). Attempts to prevent cisplatin- induced neurotoxicity may involve treatment of patients with Patients and methods nucleophilic sulfur thiols (amifostine, diethyldithiolcarbamate and others), adrenocorticotropic hormone analogs (ORG 2766) Prepar ation and character istic s of Lipoplatin. C ispla tin w as and calcium channel antagonists (19). Never the less, the clinical purchase d from H eraeus /Flavine (mw 300). The lipid s hell of relevance of the protective effect is small (20-22). A randomized Lipoplatin is c ompose d of 1,2-DiPalmitoyl-s n-G lyce ro-3- double-blind placebo-controlled study using the natural [Phospho-ra c-(1-glyc e rol)] (sodium s alt) (DPPG , mw 745, Lipoid protective agent glutathione (glutamyl-cysteinyl-glycine) in 50 G mbH), s oy phospha tidyl c holine (SPC-3, mw 790 Lipoid patients with gastric cancer undergoing cisplatin+5- G mbH), c holeste rol (CHO L, mw 386.66, A vanti Polar Lipids) FU+epirubicin+leukovorin dose intensive chemotherapy (23) a nd me thoxy-polyethylene glycol–distea royl showed reduced neuropathies in the glutathione arm. phosphatidyletha nola mine lipid conjugate (mMPEG2000-DSPE, Ototoxicity symptoms include tinnitus (ringing in the ears), mw 2807, G e nzyme ). The ratio of lipid: c ispla tin is 10.24 : 1.0 deafness and hearing difficulties especially in noisy (w/w). The content of Lipoplatin in cholesterol is 11.6% (w/w) of environments or difficulty in hearing high frequency sounds the total lipid. It is c a lcula te d that 1.3 mg cholesterol are being such as the ticking of a watch or the telephone ringing (24). The injected per mg cisplatin in its Lipoplatin formulation. most important factor predicting persistent ototoxicity was a The type of liposome particles used in Lipoplatin is a cumulative dose of over 600 mg/m2 cisplatin in testicular cancer proprietary formulation of an average size of 110 nm. Lipoplatin is being provided in 50-ml opaque glass vials of

3032 Boulikas et al: Tumor Uptake of Lipoplatin

3mg/ml (concentration refers to cisplatin). Although cisplatin is times its volume (0.2-2 ml) of saline. The sample was light-sensitive, its Lipoplatin formulation appears to be resistant centrifuged at 21,000g for 2 min in a refrigerated microfuge. to disintegration by light because liposomes shield the drug. The supernatant was assayed for platinum levels. The tissue However, as a precaution it is recommended to be stored in the pellet was further treated with one time its original volume (0.1- dark. Lipoplatin is stored at 4 0C with an expiration date of two 1.0 ml) of 10% SDS, centrifuged at 21,000g for 2 min and the years. supernatant was analyzed for platinum levels as above. In some cases, the pellet was further digested with proteinase K in the Study design. Criteria of patient selection included: presence of 10% SDS overnight at 65 oC and the supenatant was histologically confirmed type of malignancy; operable stage of analyzed for platinum levels. Platinum levels were normalized to disease; patients gave their signed informed consent. Lipoplatin mg platinum/g tissue. Standard cisplatin curves of known was used at underdose as monotherapy (100 mg/m2) known platinum concentration were used to calculate platinum levels in from previous studies not to cause adverse effects that would all extracts. interfere with the recovery of the patients or would complicate surgery (such as the ability of wound repair). The MTD of Results Lipoplatin is above 350 mg/m2 (30). The protocol was approved both by the ethics committee of Targe ting hepatocellular adenoc ar cinom a w ith Lipoplatin. A 49- the Errikos Dunant Hospital and by the Hellenic Drug year female w ith hepatoce llula r ade noc arc inoma was infused with Organization. The procedures followed were in accordance with a s ingle dose of 100 mg/m2 Lipoplatin a s s ingle a gent during a the Helsinki Declaration of the World Medical Association. period of 8h the day before surgery. There were no acute s ide e ffects as a res ult of Lipoplatin infusion. Surgery w as the option Patient characteristics . This s tudy involved four patients a t s tage decide d by her treating physic ians. During surgery specime ns III of their disea se with the following c hara c teris tics : Patient 1: A w ere removed (at a pproximate ly 18h from infusion start) from the female patient w ith hepatocellula r a denoc arcinoma. Patie nt 2: A liver tumor, the a djace nt normal liver tissue , a c olon metastatic male patient with gas tric canc e r and liver metas ta s is. Patient 3: A tumor, the adjac ent normal c olon tissue a nd c olon fat. The male patient with gas tric canc e r. Patient 4: A male patient w ith s pecimens w ere proces sed a s des cribed in Mate ria ls and M ethods c olon canc e r. a nd the platinum levels in the saline-soluble and SDS-soluble fractions w ere a ssaye d. The total platinum on the s aline-s oluble Patient evaluation and tre atment. Before study e ntry a ll pa tients material repres e nts c ispla tin that is trapped in the tissue a nd either underwent the following evalua tion: complete medica l history and has not rea cted with macromole c ules, or has formed coordination physical e xamination, tumor mea surement or evaluation, W HO c omplexe s w ith s oluble molecule s such as glutathione. Platinum performanc e s tatus , a n electroc ardiogram, a ne urological levels in the SDS-soluble fraction repres ents cisplatin bound to e xamination. Prior to Lipoplatin infusion c omplete hema tology macromolec ule s that a re not soluble in s a line extraction but are a nd s e rum c hemis try including c holes terol, triglyce ride leve ls, s olubilized by SDS rupture of macromolec ule s a nd ce ll structure s, blood urea, s erum creatinine , liver function tests and urine a s well as by SDS rupture of nuclei, c ellular membranes and a nalys is w e re pe rformed. Staging was determine d by chest a nd liposome s. Their s um gives the total platinum and w as e xpress ed a bdominal C T sca ns, a nd oc casiona l magnetic resona nce imaging a s mg platinum /g tiss ue. e ndosc opy (gastros copy) and colonosc opy. The he patoc ellular adenoc a rcinoma tissue accumulate d about 2 All patients were infused with a single dose of 100 mg/m 38 mg total platinum /g tissue c ompared with 19 mg total platinum Lipoplatin as single agent diluted in 1 L 5% dextrose during a / g normal liver tiss ue. In other words the liver tumor had uptaken period of 8h. twice as much total platinum a fter Lipoplatin infusion c ompared to adjacent normal liver tissue (Table 1). Analysis of plasma for platinum. Blood was drawn before The differenc e betwee n Lipoplatin a c cumulation in a c olon Lipoplatin infusion as well as prior to the operation and placed metastas is compare d to an adjac ent normal c olon tissue w as e ven in EDTA-containing centrifuge tubes. Blood was then more drama tic . The c olon metastas is uptook about 50 times more centrifuged to remove cells and total plasma was analyzed for Lipoplatin than a normal c olon tissue specime n (Table 1). The total platinum (i.e. free plus protein bound plus liposomal) using data demonstrate a preferentia l uptake of Lipoplatin by liver both the Perkin Elmer Analyst AA200 (flame) and Perkin Elmer tumor following intrave nous infusion c ompared to normal liver AA 700 Graphite Furnace Atomic Absorption Spectrometers tissue . There w a s a difference in the overa l a ccumula tion of the equipped with a platinum lamp. Plasma platinum levels at 20h liposoma lly e nca psula te d drug in the liver and c olon tumors; from the start of Lipoplatin infusion were ~3 mg/ml in acordance overa ll the uptake of Lipoplatin by the liver tumor w as about 6 with previous studies (30). times higher that the colon metas ta tic tumor (38.36 vers us 6.61 mg total platinum / g tissue , Table 1). Such differences c ould a rise Analys is of s urgic al specime ns for platinum. During surgery from the biodistribution properties of the drug but a lso from (performed at ~ 20h from Lipoplatin infusion s tart) specime ns difference s in vasculariz a tion betw e en the liver tumor a nd its w ere obtained (0.1-1g) from the primary or metas ta tic tumor, the c olon meta s tasis . a djace nt normal tissue, a nd in some case s from c olon fat or O ur e xtraction method using saline gives an indication of muscle tiss ue. platinum trapped in the tissue (saline -s oluble ) where as the Samples were blindly analyzed for platinum content as s ubseque nt SDS e xtrac tion indicates platinum that has e ntere d the follows. 0.1-1.0g tissue was ground and homogenized into two tumor cell and/or has reac ted with macromolec ule s. Table 1

3033 ANTICANCER RESEARCH 25: 3031-3040, (2005) ______

Table I. Platinum levels in tumor and normal tissue in a patient with T able II. P latinum le vels in liver m etastasis and normal live r tissue in a hepatocellular carcinoma and colon metastases patient with gastric cance r

Patient 1: Pt (mg/g TOTAL Pt Ratio Patient 2: Pt (mg/gr TOTAL Pt (mg Ratio Hepatocellular tissue) in (mg/g tissue) tumor Pt / tissue) in saline / gr tissue) tumor Pt / G astric cance r w saline or SDS normal Pt or SDS Normal tissue liver meta sta sis fractions fractions Pt liver tumor 5.18 Liver 34.51 saline 38.36 131.15 saline 1.96 6.26 liver tumor SDS 33.18 Liver 96.64 normal liver 16.45 metastasis SDS tissue saline 19.61 Normal liver 16.94 normal liver 3.16 tissue saline 20.94 tissue SDS colon metastasis 4.44 Normal liver 4.00 saline 6.61 tissue SDS Normal fat 8.38 49.53 saline 51.91 colon metastasis 2.17 SDS Normal fat 43.53 normal colon 0.06 SDS tissue saline 0.13 normal colon 0.08 tissue SDS respe c tively, Table 3). W e s urmized that the difference in colon fat saline 0.96 platinum levels betwee n the s a me tumor may a rise from the 1.79 colon fat SDS 0.83 differnt locations of the specime ns in the tumor mass a nd likely to relate to difference s in the va scula riza tion. This needs to be indica tes that the liver tumor contains about 5 mg platinum trapped further explored in future s tudies w ith a s imulata neous e xamination of vasculariz a tion. H owe ver, either tumor s pecime n in the tiss ue / g tissue a nd 33 mg platinum that has reacte d with displa ye d 40 and 10 times respe ctive ly more platinum c ompared macromolec ule s /g tissue. O n the c ontrary, normal liver tiss ue to normal s tomac h tissue (Table 3). Consistent w ith findings in c ontains a bout 16 mg platinum trapped in the tissue / g tiss ue a nd patients 1 and 2, the platinum trapped in tumor specime n 1 w a s 3 mg platinum that has reacte d with mac romolecule s /g tissue 42mg/g tissue vers us 220mg/g tissue of platinum that rea cted with (Table 1). macromolec ule s. Specime n 2 had 28 mg/g tissue traappe d platinum ve rsus 37 mg/g tissue that reac ted w ith macromolec ule s. Targeting a liver metastasis from a primary gastric cancer. A N ormal s tomac tissue displayed 2.6 mg/g tissue vers us 4.0 mg/g male with gastric cancer and liver metastasis (Patient 2) was treated with 100 mg/m2 Lipoplatin as single agent using an 8h tissue platinum that was trappe d versus macromolec ule -bound. infusion one day before surgery. During surgery specimens were A gain stomach tumor 1 a ppears to have about 5 times more removed from the liver metastatic tumor, the adjacent normal platinum in the form that presuma mbly rea cted with liver tissue, and fat. The liver metastatic specimen displayed a macromolec ule s a nd thus induces damage to c ells compare d to total amount of 131.15 mg platinum/ gr tissue compared to platinum s imply trapped in the tissue (220:42, Table 3) whereas 20.94 mg platinum/ gr normal liver tissue (Table 2). This gives a s pecimen 2 and normal gastric tissue has only 1.5 times more or a bout the same platinum in the two fractions. 6.26 higher level of platinum in the liver metastatic tumor Consistent with findings in Patient 2, the fat tissue in Patient compared to normal liver tissue; it is emphasized that the 3 also displayed significant levels of platinum (41 mg/g fat comparison here is between a gastric tumor in liver with liver tissue). tissue. Fat removed during surgery appears to display signifacant levels of total platinum (51.91 mg platinum/ gr tissue) Accumulation of platinum in a colon tumor after Lipoplatin supposedly relating to the lipophilic nature of the drug. infusion. A male with colon cancer was treated with 100 mg/m2 Lipoplatin as single agent during a period of 8h. During surgery Targe ting gastr ic tumor s. A male with gas tric canc e r (Patient 3) specimens were obtained from the colon tumor (in duplicate), w as treate d w ith 100 mg/m2 Lipoplatin a s s ingle a gent given the adjacent normal colon tissue, muscle and fat. Total platinum during a n infusion pe riod of 8h. During s urgery two different levels in the two colon tumor specimens were 11.26 and 7.69 mg s pecimens w ere obtained from the stomach tumor, the a djace nt platinum /g tissue compared to 0.06 mg/g of normal colon tissue normal s tomach tissue , and fat. Samples were blindly analyze d for platinum c ontent. Both specime ns of gastric tumors appea r to (Table 4). This gives 204- and 140-fold higher levels of a ccumula te the highes t amounts of platinum among all specime ns platinum between the colon tumor and normal colon tissue. a nalyz ed in this study (262.62 and 66.38 mg Pt/g tis s ue Consistent with findings in Patients 2 and 3 the fat tissue in Patient 4 also displayed significant levels of platinum (20 mg/g

3034 Boulikas et al: Tumor Uptake of Lipoplatin

Table III. Platinum levels in a gastric tumor and its adjacent normal Table IV. Platinum levels in a colon tumor, its adjacent normal colon gastric tissue. tissue, as well as normal fat and muscle tissues

Patient 3: Pt (mg/gr tissue) TOTAL Pt (mg / Ratio Patient 4: Pt (mg/gr tissue) TOTAL Pt (mg / Ratio Colon cancer in saline or SDS gr tissue) tumor Pt / Colon cancer in saline or SDS gr tissue) tumor Pt / fractions Normal Pt fractions Normal Pt Stomach tumor 42.17 colon tumor 1 4.42 1 saline 262.62 39.87 Saline 11.26 204.72 colon tumor 1 6.85 Stomach tumor 220.45 SDS 1 SDS colon tumor 2 1.86 Stomach tumor 28.46 Saline 7.69 139.70 2 saline 66.38 10.08 colon tumor 2 5.83 SDS Stomach tumor 37.92 normal colon 0.02 2 SDS tissue Saline 0.06 normal Stomach 2.62 normal colon 0.04 tissue saline 6.59 tissue SDS Muscle tissue 0.36 normal Stomach 3.97 Saline 0.36 tissue SDS Muscle tissue 0.00 Normal Fat 10.36 SDS tissue Saline 41.12 fat tissue Saline 4.34 Normal Fat 30.76 19.98 tissue SDS fat tissue SDS 15.64

/g tissue, respe ctive ly, Table 4). Following the s uggestion that fat tissue). The muscle tissue had very small amounts of Lipoplatin nanopartic le s have the intrinsic property to extrava sa te platinum (0.36 mg platinum/g tissue). through the a ltere d tumor vasc ula ture, the findings of high platinum a c cumulation in c ertain tumor s pecime ns , e specially Discussion gastric, reporte d here might c orrelate w ith the vas culariz ation of the tumor. Although fat tiss ue lacks high degree of vasc ularization High accumulation of Lipoplatin in tumors and its low toxicity. it displaye d a high a ccumula tion of total platinum in s urgica l The present study directly shows platinum accumulation in s pecimens e xcis e d from three out of four patie nts (52 mg c ispla tin tumors compared to normal tissue after intravenous infusion of /g fat tiss ue, Table 2; 41 mg /g fat tiss ue Table 3; 20 mg cis pla tin /g Lipoplatin in four cases of cancer patients. The tumor fat tissue Table 4). This might correlate to the liposomal na ture of accumulation properties of LipoplatinTM, are astonishing, and in Lipoplatin and the lipophilicity of the drug. colon tumor specimens levels that were up to 204 times higher C ispla tin, in c ombination with other drugs, is being use d as than those attained in the normal tissue were documented. The first line chemotherapy a gainst e pithelia l maligna ncies and a s PEG polymer coating used to camouflage Lipoplatin is s econd line trea tment a gains t most other advance d c ancers. suggested to be responsible for the enhanced circulation of the H oweve r, c isplatin c a uses seve re renal tubular damage , reducing drug, its ability to pass undetected by the macrophages and glomerular filtration by inducing oxidative stress in renal tubular immune cells, and its property to extravasate preferentially and cells leading to apoptotic death (10). A major dose-limiting side infiltrate solid tumors and metastases through the altered and effect of cisplatin is ototoxicity leading to hearing loss by often compromised tumor vasculature (31). Indeed, the half life inducing a poptosis in auditory s ensory c ells and destruction of of Lipolatin was found to be 60-114 h in patients compared to hair cells linked w ith tinnitus; this is proportional to the less than 6h for cisplatin (30). Futhermore, unlike other cumulative dose of cisplatin and independent of treatment liposomal drugs endowed with tumor targeting abilities such as schedules (32). Peripheral neurotoxic ity is also a dose-limiting Doxil/Caelyx or SPI-77, Lipoplatin is suggested to be endowed toxicity c a using loss of vibration s ense, paresthe s ia a nd sensory with cell membrane fusion properties because of the DPPG lipid a taxia . A cute emesis after c isplatin a dministration is mediated by molecule used in its formulation (28). s erotonin releas e from enterochroma ffin gut mucosal c ells and A mong the various surgical s pec imens e xamined, gastric s timulation of serotonin 5-HT3-re ce ptors (reviewed in 7). tumors revealed the highes t levels of total platinum (up to 263 mg This s tudy as w e ll a s a previous one (30) s how no s ide c ispla tin /g tissue, Table 3) followed by liver tumors (up to 38 mg e ffects after a single 8h-infusion of Lipoplatin at 100 mg/m2 (from c ispla tin /g tissue, Table 1). The lowes t levels of total platinum full blood analysis s even days after drug trea tment) concomitant a ccumula tion among tumor s pecimens w as observe d in a w ith higher platinum accumulation in s urgical specime ns . The metastatic colon tumor from a primary hepatoc e llular cance r (6.6 present study provides one reasonable explanation for the lower mg c ispla tin /g tissue , Table 1) a nd two primary colon tumor toxicity of the drug by showing a higher uptake by the tumor s pecimens from a colon cance r patient (11.2 and 7.7 mg cis pla tin tissue compared to normal tissue. In addition, Lipoplatin appears to be bound more in macromolecules in cancerous tissue but simply trapped and not reacted with macromolecules in

3035 ANTICANCER RESEARCH 25: 3031-3040, (2005) ______normal tissue (Tables 1-3) an observation that suggests one in liver tumor, 19.61 mg/g tissue in normal liver s ee Table 1; additional mechanism for lower toxicity of Lipoplatin compared 262.62 mg/g in gastric tumor, s ee Table 3) demonstrate a n to cisplatin. Of considerable importance is the fact that the a stonishing high level acc umulation in human tissue a fter i.v. neuro-, oto- and nephro-toxicity of Lipoplatin at doses up to 125 infusion of Lipoplatin compare d to the ma ximum level rea ched in mg/m2 are negligible (30). rat plasma or rat kidney. Coating the surface of liposomes with inert materials A Phase I pharmacokinetic study has shown that the designed to camouflage the liposome from the body’s host maximum level of platinum in plasma is 5mg/ml and is attained defense systems was shown to increase remarkably the plasma at 6h from infusion start; plasma platinum levels drop to lower longevity of liposomes, in a way similar to the erythrocyte amounts (~ 1-2 mg/ml) at 24h (30). On the contrary, platinum coated with a dense layer of carbohydrate groups to evade levels in all tumors examined are much higher than the -2 mg/ml (31,33). Lipoplatin has a coat of polyethylene plasma at 20 h (Tables 1-4). The data presented here show that a glycol (PEG), a modification used for many years to prolong the preferential uptake of Lipoplatin by tumors is established and is half-lives of biological proteins (such as enzymes and growth quite high at 20h under conditioins where the levels of platinum factors) reducing their immunogenicity (e.g. 34) or to coat in plasma drop to below 2 mg/ml. In addition, the maximum liposomes that were then able to circulate for remarkably long plasma levels of total platinum reached in human patients times after intravenous administration leading to a reduction in (5mg/ml) were much lower than the total platinum levels nonspecific uptake by the reticuloendothelial system (35-38). attained in certain tumors especially gastric cancer (262 mg/g The ma jor mecha nis m of cisplatin cytotoxicity arise s from tissue, Table 3). It will be interesting to determine how early its ta lent to induce monofunctiona l D NA a dducts as well as during infusion the preferential uptake of Lipoplatin by the intrastrand and interstrand crosslinks. Cisplatin lesions are tumor is established and for how long it persists. recognized by High Mobility Group (HMG) nonhistones and binding of HMG-domain proteins to cisplatin-modified DNA Prefe r ential intake of Lipoplatin by tumor cells ? Tables 1-3 show has been postulated to mediate the antitumor properties of the that tumor specime ns contain more SDS-extra cte d platinum drug (39,40). Cisplatin is an activator of stress-signaling c ompared to s aline-e xtrac ted platinum where as normal tissue pathways especially of the c-Jun N-terminal kinase (JNK) c ontains more s a line-extra cted platinum c ompared to SDS- family of mitogen-activated protein (MAP) kinases in a variety e xtrac ted platinum. Presumably, SDS-extracted platinum of cell types leading to apoptosis (41-44). The present study repres ents cisplatin molec ules bound to macromolec ule s w here a s suggests that alterations in signaling and apoptotic pathways will s aline-e xtrac te d platinum repre se nts unreac te d cis pla tin molecule s be more extensive in tumor cells because of the higher trappe d in the tissue or c ispla tin molecule s bound to small s oluble accumulation of the drug in tumors compared to normal tissue molecule s. This finding s uggests that a s ignificant frac tion of leading to their apoptotic death. Lipoplatin enters the liver tumor c e ll a s opposed to normal liver It will be important to de termine the prefe re ntial Lipoplatin tissue (Table 1), the liver metas ta s is from gastric c ancer compare d a ccumula tion and the absolute a mounts in mg platinum per g to normal liver tissue (Table 2) and to gastric tumor c ompared to tissue in type of tumors other than those e xamined here (liver, normal gastric tissue (Table 3). A s tatis tica lly-s ignifica nt number gastric or colon tumors). This approach w ould give important of tumor s pecime ns ne ed to be a nalyz ed be fore final c onclusions c lues on the thera pe utic e ffica cy of Lipoplatin simply from its c an be draw n. H oweve r, these preliminary s tudies s ugges t that targeting a bility and reac tion with macromole c ules to va rious the lower toxicity of Lipoplatin compare d to c ispla tin found in tumor types following intravenous a dministration. The da ta a nimals and in human trials (30) also arises from that in normal s howing a high acc umula tion in gastric ca ncer (Table 3) suggest a tissue most of the platinum appears to be trappe d in the tiss ue thera peutic potentia l of the drug in gastric c ancer to be tes ted in w here a s most of the platinum in tumors a ppears to have reacte d Phase II clinic a l trials. w ith macromolec ule s (Table s 1-3). This differenc e c ould a rise (i) Lipoplatin differs from the SPI-77 formulation of c ispla tin from a lterations in the me mbrane of the tumor cells rendering (45-48). SPI-77 administra tion did not result in tumor them more vulnerable to Lipoplatin penetration or fusion a ccumula tion (48). In addition, urinary e xcre tion of SPI-77 is only c ompared to the membrane of normal c ells. (ii) From a more a vid 2%-6% of the dose in 96h (48) c ompared to 40% of the total dose phagoc ytos is of Lipolatin by tumor c ells compare d to normal in 72 h for Lipoplatin (30). c ells; tumor cells might have higher phagoc ytotic a ctivities than A nimal s tudies have s hown the distribution of Lipoplatin in normal cells. the a nimal tissues a fter i.v. or i.p bolus injection (28,29). There fore, the time from infusion start is important in dete rmining Conclusion platinum le vels in va rious tiss ues. A fter Lipoplatin trea tment of rats a t 45 mg/Kg i.p. the ma ximum level rea ched was 23 mg The present study shows platinum accumulation in tumors platinum/ml in plasma (28) a nd 45 mg platinum/g tissue in kidney compared to normal tissue after intravenous infusion of a t about 20-40 min following i.p. injection (29). In both s tudies Lipoplatin to cancer patients. The tumor accumulation properties the le vels of platinum dropped to about 12 mg platinum/g tissue in of LipoplatinTM are astonishing reaching in certain cases levels kidney a t 24h a nd and to a bout 2 mg platinum/ml in plas ma. The that are 200 times higher than those attained in the normal dose used in rats was 18 times higher than the 100 mg/m2 dose tissue. This property of Lipoplatin adds to its value as a used here in humans. Neve rthele ss , the a bsolute platinum levels promising formulation of cisplatin against advanced cancers reache d in human tiss ue a t 24h from injec tion (38.36 mg/g tissue (30).

3036 Boulikas et al: Tumor Uptake of Lipoplatin

It is proposed that Lipoplatin able to ta rget tumors and 12. Thompson SW, Davis LE, Kornfeld M, Hilgers RD, Standefer JC. e liciting mild s ide e ffects can be a dministered in a dose-de nse Cisplatin neuropathy. Clinical, electrophysiologic, morphologic, and manner a nd thus have the potential to kill tumor c e lls more toxicologic studies. Cancer 54, 1269-1275, 1984. e ffectively in a w indow of opportunity linked to first line 13. Ozols RF, Young RC. High-dose cisplatin therapy in ovarian cancer. Semin Oncol 12, 21-30, 1985. c hemothe ra py before c hemores is tant c ells appea r. Acquired 14. Cersosimo RJ. Cisplatin neurotoxicity. Cancer Treat Rev 16, 195- chemoresistance of tumors is largely responsible for the very 211, 1989. discouraging response rates in second or third line chemotherapy 15. Harmers FP, Gispen WH, Neijt JP. Neurotoxic side-effects of treatments compared to first line treatments. The impact of dose- cisplatin. Eur J Cancer 27:372-376, 1991. dense Lipoplatin treatment allowing targeting of the tumor in a 16. Hovestadt A, van der Burg ME, Verbiest HB, van Putten WL, manner minimizing the likelihood of appearance of Vecht CJ. The course of neuropathy after cessation of cisplatin chemoresistant cells needs to be tested in clinical trials. Also, the treatment, combined with Org 2766 or placebo. J Neurol. 239:143-146, preferential tumor targeting of Lipoplatin causing a higher 1992. damage to the cancer tissue compared to most other tissues in 17. Bissett D, Kunkeler L, Zwanenburg L, Paul J, Gray C, Swan IR, Kerr DJ, Kaye SB. Long-term sequelae of treatment for testicular germ the body of the patient is promising by minimizing the side cell tumours. Br J Cancer 62:655-659, 1990. effects. 18. Gregg RW, Molepo JM, Monpetit VJ, Mikael NZ, Redmond D, The preliminary clinical studies reported here provide a first Gadia M, Stewart DJ. Cisplatin neurotoxicity: the relationship between evidence that the lower toxicity of the drug arises in part from its dosage, time, and platinum concentration in neurologic tissues, and tumor targeting properties; because of this Lipoplatin morphologic evidence of toxicity. J Clin Oncol 10:795-803, 1992. combinations with other drugs merits a careful assessment. 19. Alberts DS, Noel JK. Cisplatin-associated neurotoxicity: can it be LipoplatinTM is currently under Phase II and PhaseIII clinical prevented? Anticancer Drugs 6:369-383, 1995. evaluations. 20. Gandara DR, Nahhas WA, Adelson MD, Lichtman SM, Podczaski ES, Yanovich S, Homesley HD, Braly P, Ritch PS, Weisberg SR, et al. Randomized placebo-controlled multicenter evaluation of Acknowledgements diethyldithiocarbamate for chemoprotection against cisplatin-induced We are grateful to John Papadimitriou (surgeon, Errikos Dynan toxicities. J Clin Oncol 13:490-496, 1995. Hospital, Athens) for the tumor specimens and to Ioannis 21. 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