CANCER BIOTHERAPY & RADIOPHARMACEUTICALSVolume 15, Number 3, 2000Mary Ann Liebert, Inc.

Neutron Activation of NDDP, a Liposomal PlatinumAntitumor Agent

Donald S. MacLean,1 Abdul R. Khokhar,2 and Roman Perez-Soler1,3

1Department of Thoracic/Head and Neck Medical Oncology, and 2Department of ClinicalInvestigation, The University of Texas M.D. Anderson Cancer Center, Houston, Texas; 3KaplanComprehensive Cancer Center, New York University School of Medicine, New York University, New York

Cis-bis-neodecanoato-trans-R,R-1,2-diamminocyclohexane platinum (II) [NDDP] is a liposome-entrappedplatinum compound currently, in phase II clinical trials, that has been shown to undergo intraliposomalactivation. The objective of this study was to determine the feasibility of activating NDDP and using theinduced radioactivity to monitor NDDP distribution and penetration. Methods: Neutron activation analy-sis (NAA) was done on NDDP using the nuclear reactor at Texas A & M University, College Station,Texas. After a 3-hour irradiation, the NDDP samples were analyzed using an HPGE (high purity ger-manium) detector to determine the activation of the radioisotopic platinum. This was followed by HPLC-UV analysis to determine the stability of NDDP after exposure to the reactor’s core. Results: Platinumradioisotope s were produced along with potassium-40 and sodium-24. Irradiation did not result in anysignificant degradation of NDDP. Conclusions: (1) Irradiating fully synthesized NDDP is feasible for di-agnostic use if a purification step is taken after the irradiation, and (2) radiation exposure is lessened byirradiating NDDP after synthesis rather than starting with high-specific-activity isotopes.

Key words: liposome, drug stability, neutron activation analysis, lipophilic platinum complex

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INTRODUCTION

Efforts to develop less toxic non-cross-resistantanalogs of cisplatin have led to the developmentof a lipophilic cisplatin analog, cis-bis-neode-canoato-trans-R,R-1,2-diamminocyclohexaneplatinum (II) [NDDP], which is delivered in a li-posome carrier (L-NDDP). In a previously re-ported phase I study of intravenously adminis-trated L-NDDP, myelosuppression, particularlygranulocytopenia, was found to be the dose-lim-iting toxicity, and no nephrotoxicity was ob-served. Because of NDDP’s favorable depot

pharmacokinetics and lack of systemic toxicitywhen used intracavitarily1–6 we are currently per-forming two phase II clinical trials with L-NDDPadministered intrapleurally and intraperitoneally,respectively.

NDDP belongs to the family of diaminocyclo-hexane (DACH)-platinum (II) compounds. Inter-est in this class of compounds has been renewedby the very promising antitumor activity of ox-aliplatin, another DACH compound, in combina-tion with 5-fluorouracil in advanced colorectalcarcinoma.8–10 The DACH compounds have acyclohexane group attached to their aminogroups, which abrogates their cross-resistanceand reduces their nephrotoxic ity. The firstDACH-Pt compound studied was DACH-Pt-Cl2.Because this compound was insoluble in waterand most organic solvents it was not developedfurther. Alternatively, NDDP is a DACH-Pt com-

Address reprint request to Donald S. MacLean, Depart-ment of Pharmaceutical Chemistry, School of Pharmacy,The University of Kansas, 2099 Constant Avenue,Lawrence, KS 66047. Tel: 785-864-3010; Fax: 785-864-5736.

pound that was designed to be delivered in a mul-tilamellar liposome by substituting the chloridefor neodecanoato leaving groups, which resultsin an enhanced affinity for biological membranes.Free NDDP is devoid of in-vivo antitumor activ-ity.11 In the current L-NDDP formulation, the li-posome is composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in a 7:3 molar ratio and thelipid-to-NDDP ratio is 15:1, whose acidic phos-pholipid DMPG component is essential for anti-tumor activity.12 The resulting multilamellar li-posome measures 1–3 m m in diameter and actsas a vehicle and slow-release system for the drug.

The organ distribution of platinum species istypically determined by first collecting fluids ortissues and then subjecting them to atomic ab-sorption (AAS) for elemental analysis or to liq-uid scintillation counting (LSC). Although othertechniques are available, such as X-ray fluores-cence (XRF) and inductively coupled plasmamass spectrometry (ICP-MS), their use is limitedby, either insufficient detection limits (in the caseof XRF), or expense and labor (in the case ofICP-MS). Neutron activation analysis (NAA) hasthe best detection limits among assays for plat-inum. The comparative platinum limits for thevarious methodologies are 0.08 m g/mL for ICP-MS, 20 m g/mL for inductively coupled plasma-optical emission spectrometry (ICP-OES), 4m g/mL for graphite furnace AAS (GFAAS), 100m g/mL for flame AAS (FAAS)13, 0.005 m g/mLfor NAA, and 50000 m g/mL for XRF.

According to the literature, most assays forplatinum radioisotopes start with isotopicallypure Pt-195m or with Pt-191. The enriched formof Pt is then processed into the synthesized com-pound of interest. Akaboshi et al.14 used enrichedPt-195m-radiolabelled trans and cis forms of diaminodichloroplatinum (II) to compare thekilling efficiency of the two isomers in HeLacells. Hoeschele et al.15 intravenously injectedenriched Pt-195m -labeled cis and trans dichloro-diammine platinum (II) to study the tissue distri-bution. Afterward, the organs were harvested andanalyzed by LSC. LSC was also used by Desi-mone et al.16 to determine the distribution of Pt-195m-labeled cisplatin. Bénard et al.17 also usedPt-195m-labeled cisplatin, but opted for autora-diography to determine the tissue distribution.Robins et al.18 injected accelerator produced Pt-191 ethylenediamine dichloride platinum (II) intorats and assessed its activity using LSC.

In this work, we used NAA to activate the plat-

inum atom in NDDP in order to determine thefeasibility of using activated NDDP for penetra-tion and distribution studies. After spectral analy-sis of NDDP, the stability of NDDP powder wasaccessed by HPLC.

MATERIALS AND METHODS

Preparation of NDDP for Irradiation

NDDP was synthesized as previously de-scribed.19,20 Briefly, the lipophilic ligand wasprepared by starting with neodecanoic acid andpotassium hydroxide. The resulting potassiumneodecanoate was then treated with silver nitrateto yield silver neodecanoate. Separately, potas-sium platinum (IV) tetrachloride was reacted withdiaminocyclohexane (DACH), and then with sil-ver sulfate to yield cis-aquosulfato(DACH)plat-inum(II). This was reacted with sodium iodide toyield cis-(DACH)platinum(II) diiodide. cis-(DACH)platinum(II) diiodide was then combinedwith the silver neodecanoato to yield NDDP, alsoknown as cis-bis-neodecanoato-trans-R,R-1,2-di-amminocyclohexane platinum (II) [NDDP]. Thestructure of NDDP is shown in Figure 1 and 2.

Approximately 2 mg of an NDDP powder wasplaced into high-purity quart vials [HeraeusAmersil, Duluth, GA] and heat sealed. The sam-ples were then shipped to the Texas A & M Nu-clear Science Center (College Station, Texas) forirradiation in its nuclear reactor.

Irradiation and Analysis

At Texas A & M, the vials of samples were ir-radiated for 3 hours at a thermal flux of 7 E 12neutrons/ sec-cm2. The samples were allowed todecay for 5 days, and then counted for 1 hour us-ing a HPGE detector. The samples were then an-alyzed by HPLC-UV.

For HPLC-UV analysis the vials were openedand a portion of the NDDP was dissolved in 90:10methanol / water. A 50 m L (3.2 m g NDDP) sam-ple was injected. HPLC (Shimadzu, Japan) wasdone on a C-18 m -Bondapak column (WatersChromatography, Milford, MA), 90:10 methanol/water (Sigma-Aldrich, Milwaukee, WI) as eluantat a flow rate of 1.00 mL / min. UV analysis wasdone using an UV detector (Shimadzu, Japan) setat a wavelength of 224 nm as previously de-scribed.6 Thirty second fractions were collectedand analyzed on a liquid scintillation counter(Beckman, Fullerton, CA). Finally, the output of

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the column was collected for total platinumanalysis by graphite tube atomic absorption(AAS) (Varian, model GTA-96, Walnut Creek,CA) using 1200°C as the ashing temperature and2700 °C as the atomizing temperature. An unir-radiated sample was subjected to HPLC as a con-trol.

RESULTS

Elemental Analysis

Upon analysis by HPGE, various radioisotopesof platinum could be seen, along with potassiumand sodium. Figure 3 shows the spectrum of ac-tivated NDDP counted for 1 hour on an HPGEdetector after a 3 hour irradiation and a 5 day de-cay period. Silver and iodine may have been pre-sent, but were not seen at 5 days because eitherthey were minor constituents or they decayedaway prior to analysis at 5 days.

Stability of NDDP Powder

The heat of the reactor core caused a partialbreakdown of NDDP, as evidenced by a colorchange of NDDP from white to tan. The initialHPLC data appeared to indicate that 65% of theNDDP had degraded away during irradiation,shipping, and decay prior to the analysis (NDDPelutes at 8.5–9.0 minutes). Fractions collectedand analyzed by AAS gave only 33% of the plat-inum signal expected, indicating that water mayhave worked its way into the supposedly sealedvials during irradiation.

DISCUSSION

The objective of our study was to determine thefeasibility of radioisotopically activating the plat-inum-containing compound NDDP, a DACH-platinum compound currently undergoing clini-cal studies as part of a liposomal formulation.

All currently used platinum anticancer agentsundergo intracellular activation by acidic hydrol-ysis, loss of the leaving groups, and formation ofaquated species, which are thought to be the ac-

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Figure 1. Chemical synthesis scheme for the production of NDDP.

Figure 2. Progression of chemical structures from cis-platin to NDDP.

tive intermediates that lead to the formation ofPt-DNA adducts. Compounds like cisplatin orDACH-Pt-Cl2, which have chlorides as leavinggroups, are protected in the blood during circu-lation because of the high concentration of chlo-ride in plasma. However, once the compoundsenter the cell intact, their chloride groups are eas-ily displaced as a result of the very low intracel-lular concentration of chloride, and the aquatedspecies are formed. Because of their charged na-ture, it is unlikely that aquated species formed inthe extracellular space may enter the cells. Onceinside the cell, DACH-Pt-Cl2 is transformed intomonoaquo intermediates with a half-life of 1.5-5hours.6,21,22 In contrast to the case in plasma, in-tracellular biotransformation of DACH-Pt-Cl2

and trans-DACH-Pt-malonate into aquo interme-

diates takes much less time (12–15 and 21–28minutes, respectively23), and the activationreaches a plateau at about 5 hours, when 75–85%of compounds are in the active form.24

As our previous work showed, leakage of plat-inum species from the liposomes is minimal dur-ing the first hours after reconstitution,1,6 thus in-dicating that the activated aquated species remainbound to the liposomes. The total cellular plat-inum uptake after incubation of cells with L-NDDP is about 3 to 7 fold higher than after in-cubation with an equimolar concentration ofcisplatin. Therefore, all available evidence sup-ports the hypothesis that NDDP is intraliposo-mally activated into aquated DACH-Pt speciesand that these activated species are directly in-ternalized within the cell as a result of liposome-

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Figure 3. Spectrum of 4 mg of activated NDDP counted for 1 hour on an HPGE detector after 3 hours of irradiation and a 5-day decay period. (Top) 50-225 keV region indicating the abundant emission of X-rays by iridium, platinum , gold, and mercury.(Bottom) 50-600 keV region, indicating the abundant emission of photons by platinum -191.

cell membrane fusion. Accordingly L-NDDPwould not need to be activated intracellularly toexert its effect, unlike all other DACH-Pt com-pounds. This may be important in cases where

resistance to platinum compounds may be sec-ondary to reduced or slow intracellular drug ac-tivation.

Currently, abundant natural isotopic platinum

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Table 1. Neutron activation of platinum: Anticipated activities (kBq/second) of 1 mg ofNDDP after 3 hours of irradiation and its decay. 199-Pt gave the greatest activity at time0. Only 191-Pt, 193m-Pt, 195m-Pt, and 197-Pt were useful for our studies

Actualactivity at

Isotope Half-life Time 0 24 hours 5 days 10 days 5 days

191-Pt 2.96 days 2910 2310 904 280 8.88193m-Pt 4.33 days 198 168 88.8 39.9 356193-Pt 50 years 0.474 0.474 0.474 0.474 —195m-Pt 4.02 days 886 745 374 158 37.4197m-Pt 95.4 min 11700 0.333 0 0 139197-Pt 18.3 hours 25700 10400 273 2.9 109.5199m-Pt 13.5 sec 27300 0 0 0 —199-Pt 30.8 min 339000 0 0 0 —191m-Ir 4.93 sec 2910 2310 904 280 —193m-Ir 10.6 days 0.474 0.474 0.474 0.474 —199-Au 3.2 days 0 1870 722 256 184199m-Hg 42.6 min 0 1870 722 256 204

Anticipated Activity

Table 2. Isotopes seen and not seen from the irradiation of NDDP

Isotopes Energy Decayed Isotopes with Isotope not seen becauseseen (keV) isotopes low potential decayed or minor

Na-24 1368.6 Pt-197 Pt-193 Ag-108mK-40 1460 Pt-197m Ag-108

Pt-19m Ag-110mPt-191 82.4 Pt-199m Ag-110

96.3 I-128129.4172.2351.2368.7409456.5539624.1

Pt-193m 135.5Pt-195m 98.9

129.8Pt-197 191.4Ir-191m 129.4Au-199 158.4

208.2Hg-199m 129.4

K X-rays of Pt, Ir, Hg at 60 to 80 keV also seen from the decay of the numerous platinumspecies.

For Na-24 the 2754 keV was not seen; upper threshold of detector set at 2000 keV

is used in the synthesis of NDDP and activationprovides a means to determine the distributionand penetration of L-NDDP in-vivo. However,the actual activity we observed in the presentstudy was less than anticipated, and will neverreach the activity levels of enriched Pt-191 or Pt-195m. The reasons for this could be 1) a lowerneutron flux than calculated, or 2) a less-than-fullthermalization of the neutrons resulting in crosssections smaller than those used to estimate theactivity. Even with the lower than anticipated ac-tivities for platinum, activated platinum wouldcontain sufficient activity for imaging use. In ad-dition, enriched platinum Pt-191, or Pt-195mNDDP would require dilution with nonradioac-tive NDDP to obtain a sufficient amount of ma-terial to perform the experiment.

For real-time noninvasive imaging, gammascintigraphy (SPECT) works best with photon en-ergies in the range of 150–200 keV. The mostsuitable photons are those resulting from the de-cay of Pt-191, though others from Pt-195m, andIr-191m are also present. In a SPECT analysis (at24–48 hours postirradiation), Pt-197 would con-tribute significantly to the imaging, and the highlevel of X-rays would significantly contribute toimaging from all isotopes. For autoradiography,charge particle emission is best. Betas are emit-ted from Au-199, Pt-199, and Pt-197. The betasfrom Pt-199, however, are unusable because thehalf-life of Pt-199 is 30.8 minutes, too short forany autoradiograph or LSC experiments. Theutility of Pt-197 is marginal and depends on pro-cessing time; the high number of emitted X-rayswould contribute significantly to autoradiogra-phy. Alternatively, NAA of NDDP provides fora mixture of both photons and charged particles.In the 5 day interval before spectrographic analy-sis in the present study, Pt-197m, Pt-199m, andPt-199 decayed away. However, in an actual au-toradiography study, these isotopes would not beexpected to contribute to the imaging, while theirdaughters (such as Au-199) might. Satisfactoryautoradiography would require a 5-day wait fromirradiation to the start of autoradiography. At notime would the activity from Pt-193 be expectedto contribute to either process. Table 1 showsthere is about a 3 fold difference between the ex-pected activities of irradiated NDDP and the mea-sured activities.

Our NDDP sample may have contained silveror iodine impurities, but the handling and decaytimes would not have allowed these to contributeto the result of imaging and distribution studies.

In addition, because of their high energies andlow activities, sodium-24 and potassium-40would be expected to clear quickly when admin-istered and thus, not contribute to the scinti-graphic image (Table 2).

CONCLUSION

In summary, the color change of NDDP observedin our study suggests that irradiated NDDP mightrequire purification if it is to be used diagnosti-cally. Neutron-activated NDDP results in a lowerspecific activity than achieved by synthesizingNDDP from isotopically pure Pt-191 or Pt-195m,but the radiation exposure to personnel is sub-stantially less. Thus once NDDP is incorporatedinto liposomes and reconstituted in 0.9% sodiumchloride, L-NDDP can be used as an effective de-livery system for the activated platinum species.The radioactivity can then be used to determinepenetration and distribution by either autoradi-ography or SPECT. Future studies will focus onimproving the penetration of L-NDDP into cells,determining the organ distribution of L-NDDP,identifying the intermediates of NDDP activationusing 3-H labeled NDDP, and fully characteriz-ing the chemical species that are formed after L-NDDP reconstitution by using in-line mass spec-troscopy.

ACKNOWLEDGMENTS

These studies were supported by NCI Grant CA60496 and by Aronex Pharmaceuticals (TheWoodlands, TX 77381).

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