CANCER BIOTHERAPY & RADIOPHARMACEUTICALS

Volume 15, Number 3, 2000

Mary Ann Liebert, Inc.

Neutron Activation of NDDP, a Liposomal Platinum

Antitumor Agent
Donald S. MacLean, 1 Abdul R. Khokhar, 2 and Roman Perez-Soler 1,3
1
Department of Thoracic/Head and Neck Medical Oncology, and 2 Department of Clinical
Investigation, The University of Texas M.D. Anderson Cancer Center, Houston, Texas; 3 Kaplan
Comprehensive 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-entrapped

platinum compound currently, in phase II clinical trials, that has been shown to undergo intraliposom al
activation. The objective of this study was to determine the feasibility of activating NDDP and using the
induced radioactivity to monitor NDDP distribution and penetration. Methods: Neutron activation analysis (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 germanium) detector to determine the activation of the radioisotopic platinum. This was followed by HPLCUV analysis to determine the stability of NDDP after exposure to the reactors core. Results: Platinum
radioisotope s were produced along with potassium-40 and sodium-24. Irradiation did not result in any
significant degradation of NDDP. Conclusions: (1) Irradiating fully synthesized NDDP is feasible for diagnostic use if a purification step is taken after the irradiation, and (2) radiation exposure is lessened by
irradiating NDDP after synthesis rather than starting with high-specific-activity isotopes.
Key words: liposom e, drug stability, neutron activation analysis, lipophilic platinum complex
INTRODUCTION
Efforts to develop less toxic non-cross-resistant
analogs of cisplatin have led to the developm ent
of a lipophilic cisplatin analog, cis-bis-neodecanoato-tran s-R,R-1,2-diamminocyclohexane
platinum (II) [NDDP], which is delivered in a liposome carrier (L-NDDP). In a previously reported phase I study of intravenously administrated L-NDDP, myelosuppression, particularly
granulocytopenia, was found to be the dose-limiting toxicity, and no nephrotoxicity was observed. Because of NDDPs favorable depot
Address reprint request to Donald S. MacLean, Department of Pharmaceutical Chemistry, School of Pharmacy,
The University of Kansas, 2099 Constant Avenue,
Lawrence, KS 66047. Tel: 785-864-3010; Fax: 785-8645736.

pharmacokinetics and lack of systemic toxicity

when used intracavitarily 1 6 we are currently performing two phase II clinical trials with L-NDDP
administered intrapleurally and intraperitoneally,
respectively.
NDDP belongs to the family of diaminocyclohexane (DACH)-platinum (II) compounds. Interest in this class of compounds has been renewed
by the very promising antitumor activity of oxaliplatin, another DACH compound, in combination with 5-fluorouracil in advanced colorectal
carcinoma.8 10 The DACH compounds have a
cyclohexane group attached to their amino
groups, which abrogates their cross-resistance
and reduces their nephrotoxic ity. The first
DACH-Pt compound studied was DACH-Pt-Cl2 .
Because this compound was insoluble in water
and most organic solvents it was not developed
further. Alternatively, NDDP is a DACH-Pt com253

pound that was designed to be delivered in a multilamellar liposome by substituting the chloride
for neodecanoato leaving groups, which results
in an enhanced affinity for biological membranes.
Free NDDP is devoid of in-vivo antitumor activity. 11 In the current L-NDDP formulation, the liposome is composed of dimyristoylphosphatidyl
choline (DMPC) and dimyristoylphosphatidyl
glycerol (DMPG) in a 7:3 molar ratio and the
lipid-to-NDDP ratio is 15:1, whose acidic phospholipid DMPG component is essential for antitumor activity. 12 The resulting multilamellar liposome measures 13 m m in diameter and acts
as a vehicle and slow-release system for the drug.
The organ distribution of platinum species is
typically determined by first collecting fluids or
tissues and then subjecting them to atomic absorption (AAS) for elemental analysis or to liquid scintillation counting (LSC). Although other
techniques are available, such as X-ray fluorescence (XRF) and inductively coupled plasma
mass spectrometry (ICP-MS), their use is limited
by, either insufficient detection limits (in the case
of XRF), or expense and labor (in the case of
ICP-MS). Neutron activation analysis (NAA) has
the best detection limits among assays for platinum. The comparative platinum limits for the
various methodologies are 0.08 m g/mL for ICPMS, 20 m g/mL for inductively coupled plasmaoptical emission spectrometry (ICP-OES), 4
m g/mL for graphite furnace AAS (GFAAS), 100
m g/mL for flame AAS (FAAS) 13 , 0.005 m g/mL
for NAA, and 50000 m g/mL for XRF.
According to the literature, most assays for
platinum radioisotopes start with isotopically
pure Pt-195m or with Pt-191. The enriched form
of Pt is then processed into the synthesized compound of interest. Akaboshi et al.14 used enriched
Pt-195m-radiolabelled trans and cis forms of
diaminodichlorop latinum (II) to compare the
killing efficiency of the two isomers in HeLa
cells. Hoeschele et al.15 intravenously injected
enriched Pt-195m -labeled cis and trans dichlorodiammine platinum (II) to study the tissue distribution. Afterward, the organs were harvested and
analyzed by LSC. LSC was also used by Desimone et al.16 to determine the distribution of Pt195m-labeled cisplatin. Bnard et al.17 also used
Pt-195m-labeled cisplatin, but opted for autoradiography to determine the tissue distribution.
Robins et al.18 injected accelerator produced Pt191 ethylenediamine dichloride platinum (II) into
rats and assessed its activity using LSC.
In this work, we used NAA to activate the plat254

inum atom in NDDP in order to determine the

feasibility of using activated NDDP for penetration and distribution studies. After spectral analysis of NDDP, the stability of NDDP powder was
accessed by HPLC.
MATERIALS AND METHODS
Preparation of NDDP for Irradiation
NDDP was synthesized as previously described. 19,20 Briefly, the lipophilic ligand was
prepared by starting with neodecanoic acid and
potassium hydroxide. The resulting potassium
neodecanoate was then treated with silver nitrate
to yield silver neodecanoate. Separately, potassium platinum (IV) tetrachloride was reacted with
diaminocyclohexane (DACH), and then with silver sulfate to yield cis-aquosulfato(DACH)platinum(II). This was reacted with sodium iodide to
yield cis-(DACH)platinum(II) diiodide. cis(DACH)platinum(II) diiodide was then combined
with the silver neodecanoato to yield NDDP, also
known as cis-bis-neodecanoato-trans-R,R-1,2-diamminocyclohexane platinum (II) [NDDP]. The
structure of NDDP is shown in Figure 1 and 2.
Approxim ately 2 mg of an NDDP powder was
placed into high-purity quart vials [Heraeus
Amersil, Duluth, GA] and heat sealed. The samples were then shipped to the Texas A & M Nuclear Science Center (College Station, Texas) for
irradiation in its nuclear reactor.
Irradiation and Analysis
At Texas A & M, the vials of samples were irradiated for 3 hours at a thermal flux of 7 E 12
neutrons/ sec-cm 2 . The samples were allowed to
decay for 5 days, and then counted for 1 hour using a HPGE detector. The samples were then analyzed by HPLC-UV.
For HPLC-UV analysis the vials were opened
and a portion of the NDDP was dissolved in 90:10
methanol / water. A 50 m L (3.2 m g NDDP) sample was injected. HPLC (Shimadzu, Japan) was
done on a C-18 m -Bondapak column (Waters
Chromatography, Milford, MA), 90:10 methanol/
water (Sigma-Aldrich, Milwaukee, WI) as eluant
at a flow rate of 1.00 mL / min. UV analysis was
done using an UV detector (Shimadzu, Japan) set
at a wavelength of 224 nm as previously described. 6 Thirty second fractions were collected
and analyzed on a liquid scintillation counter
(Beckman, Fullerton, CA). Finally, the output of

Figure 1.

Chemical synthesis scheme for the production of NDDP.

the column was collected for total platinum

analysis by graphite tube atomic absorption
(AAS) (Varian, model GTA-96, Walnut Creek,
CA) using 1200C as the ashing temperature and
2700 C as the atomizing temperature. An unirradiated sample was subjected to HPLC as a control.

RESULTS

DISCUSSION
The objective of our study was to determine the
feasibility of radioisotopically activating the platinum-containing compound NDDP, a DACHplatinum compound currently undergoing clinical studies as part of a liposomal formulation.
All currently used platinum anticancer agents
undergo intracellular activation by acidic hydrolysis, loss of the leaving groups, and formation of
aquated species, which are thought to be the ac-

Elemental Analysis
Upon analysis by HPGE, various radioisotopes
of platinum could be seen, along with potassium
and sodium . Figure 3 shows the spectrum of activated NDDP counted for 1 hour on an HPGE
detector after a 3 hour irradiation and a 5 day decay period. Silver and iodine may have been present, but were not seen at 5 days because either
they were minor constituents or they decayed
away prior to analysis at 5 days.
Stability of NDDP Powder
The heat of the reactor core caused a partial
breakdown of NDDP, as evidenced by a color
change of NDDP from white to tan. The initial
HPLC data appeared to indicate that 65% of the
NDDP had degraded away during irradiation,
shipping, and decay prior to the analysis (NDDP
elutes at 8.59.0 minutes). Fractions collected
and analyzed by AAS gave only 33% of the platinum signal expected, indicating that water may
have worked its way into the supposedly sealed
vials during irradiation.

Figure 2. Progression of chemical structures from cisplatin to NDDP.

255

Figure 3. Spectrum of 4 mg of activated NDDP counted for 1 hour on an HPGE detector after 3 hours of irradiation and a 5day 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.

tive intermediates that lead to the formation of

Pt-DNA adducts. Compounds like cisplatin or
DACH-Pt-Cl2 , which have chlorides as leaving
groups, are protected in the blood during circulation because of the high concentration of chloride in plasma. However, once the compounds
enter the cell intact, their chloride groups are easily displaced as a result of the very low intracellular concentration of chloride, and the aquated
species are formed. Because of their charged nature, it is unlikely that aquated species formed in
the extracellular space may enter the cells. Once
inside the cell, DACH-Pt-Cl2 is transformed into
monoaquo intermediates with a half-life of 1.5-5
hours. 6,21,22 In contrast to the case in plasma, intracellular biotransformation of DACH-Pt-Cl2
and trans-DACH-Pt-malonate into aquo interme256

diates takes much less time (1215 and 2128

minutes, respectively23 ), and the activation
reaches a plateau at about 5 hours, when 7585%
of compounds are in the active form.24
As our previous work showed, leakage of platinum species from the liposom es is minimal during the first hours after reconstitution,1,6 thus indicating that the activated aquated species remain
bound to the liposomes. The total cellular platinum uptake after incubation of cells with LNDDP is about 3 to 7 fold higher than after incubation with an equimolar concentration of
cisplatin. Therefore, all available evidence supports the hypothesis that NDDP is intraliposomally activated into aquated DACH-Pt species
and that these activated species are directly internalized within the cell as a result of liposome-

Table 1. Neutron activation of platinum: Anticipated activities (kBq/second) of 1 mg of

NDDP after 3 hours of irradiation and its decay. 199-Pt gave the greatest activity at time
0. Only 191-Pt, 193m -Pt, 195m-Pt, and 197-Pt were useful for our studies

Half-life

Time 0

24 hours

5 days

10 days

Actual
activity at
5 days

2.96 days
4.33 days
50 years
4.02 days
95.4 min
18.3 hours
13.5 sec
30.8 min
4.93 sec
10.6 days
3.2 days
42.6 min

2910
198
0.474
886
11700
25700
27300
339000
2910
0.474
0
0

2310
168
0.474
745
0.333
10400
0
0
2310
0.474
1870
1870

904
88.8
0.474
374
0
273
0
0
904
0.474
722
722

280
39.9
0.474
158
0
2.9
0
0
280
0.474
256
256

8.88
356

37.4
139
109.5

184
204

Anticipated Activity
Isotope
191-Pt
193m-Pt
193-Pt
195m-Pt
197m-Pt
197-Pt
199m-Pt
199-Pt
191m-Ir
193m-Ir
199-Au
199m-Hg

cell membrane fusion. Accordingly L-NDDP

would not need to be activated intracellularly to
exert its effect, unlike all other DACH-Pt compounds. This may be important in cases where
Table 2.

Isotopes seen and not seen from the irradiation of NDDP

Isotopes
seen

Energy
(keV)

Decayed
isotopes

Na-24
K-40

1368.6
1460

Pt-191

82.4
96.3
129.4
172.2
351.2
368.7
409
456.5
539
624.1
135.5
98.9
129.8
191.4
129.4
158.4
208.2
129.4

Pt-197
Pt-197m
Pt-19m
Pt-199m

Pt-193m
Pt-195m
Pt-197
Ir-191m
Au-199
Hg-199m

resistance to platinum compounds may be secondary to reduced or slow intracellular drug activation.
Currently, abundant natural isotopic platinum

Isotopes with
low potential
Pt-193

Isotope not seen because

decayed or minor
Ag-108m
Ag-108
Ag-110m
Ag-110
I-128

K X-rays of Pt, Ir, Hg at 60 to 80 keV also seen from the decay of the numerous platinum
species.
For Na-24 the 2754 keV was not seen; upper threshold of detector set at 2000 keV

257

is used in the synthesis of NDDP and activation

provides a means to determine the distribution
and penetration of L-NDDP in-vivo. However,
the actual activity we observed in the present
study was less than anticipated, and will never
reach the activity levels of enriched Pt-191 or Pt195m. The reasons for this could be 1) a lower
neutron flux than calculated, or 2) a less-than-full
thermalization of the neutrons resulting in cross
sections smaller than those used to estimate the
activity. Even with the lower than anticipated activities for platinum, activated platinum would
contain sufficient activity for imaging use. In addition, enriched platinum Pt-191, or Pt-195m
NDDP would require dilution with nonradioactive NDDP to obtain a sufficient amount of material to perform the experiment.
For real-time noninvasive imaging, gamma
scintigraphy (SPECT) works best with photon energies in the range of 150200 keV. The most
suitable photons are those resulting from the decay of Pt-191, though others from Pt-195m, and
Ir-191m are also present. In a SPECT analysis (at
2448 hours postirradiation), Pt-197 would contribute significantly to the imaging, and the high
level of X-rays would significantly contribute to
imaging from all isotopes. For autoradiography,
charge particle emission is best. Betas are emitted from Au-199, Pt-199, and Pt-197. The betas
from Pt-199, however, are unusable because the
half-life of Pt-199 is 30.8 minutes, too short for
any autoradiograph or LSC experiments. The
utility of Pt-197 is marginal and depends on processing time; the high number of emitted X-rays
would contribute significantly to autoradiography. Alternatively, NAA of NDDP provides for
a mixture of both photons and charged particles.
In the 5 day interval before spectrographic analysis in the present study, Pt-197m, Pt-199m, and
Pt-199 decayed away. However, in an actual autoradiography study, these isotopes would not be
expected to contribute to the imaging, while their
daughters (such as Au-199) might. Satisfactory
autoradiography would require a 5-day wait from
irradiation to the start of autoradiography. At no
time would the activity from Pt-193 be expected
to contribute to either process. Table 1 shows
there is about a 3 fold difference between the expected activities of irradiated NDDP and the measured activities.
Our NDDP sample may have contained silver
or iodine impurities, but the handling and decay
times would not have allowed these to contribute
to the result of imaging and distribution studies.
258

In addition, because of their high energies and

low activities, sodium-24 and potassium -40
would be expected to clear quickly when administered and thus, not contribute to the scintigraphic image (Table 2).
CONCLUSION
In summary, the color change of NDDP observed
in our study suggests that irradiated NDDP might
require purification if it is to be used diagnostically. Neutron-activated NDDP results in a lower
specific activity than achieved by synthesizing
NDDP from isotopically pure Pt-191 or Pt-195m,
but the radiation exposure to personnel is substantially less. Thus once NDDP is incorporated
into liposomes and reconstituted in 0.9% sodium
chloride, L-NDDP can be used as an effective delivery system for the activated platinum species.
The radioactivity can then be used to determine
penetration and distribution by either autoradiography or SPECT. Future studies will focus on
improving the penetration of L-NDDP into cells,
determining the organ distribution of L-NDDP,
identifying the intermediates of NDDP activation
using 3-H labeled NDDP, and fully characterizing the chemical species that are formed after LNDDP reconstitution by using in-line mass spectroscopy.
ACKNOWLEDGMENTS
These studies were supported by NCI Grant CA
60496 and by Aronex Pharmaceuticals (The
Woodlands, TX 77381).
REFERENCES
1. Han I, Khokhar AR, Perez-Soler R. Intraliposomal conversion of lipophilic cis-bis-carboxylato-trans-R, R-1,2diaminocyclohexane-platinum (II) complexes into cis-bisdichloro-trans-R, R-1, 2-diaminocyclohexane-platinum
(II). Cancer Chemother Pharmacol 1996; 39 (12): 17.
2. Khokhar AR, Al-Baker S, Brown T, Perez-Soler R.
Chemical and biological studies on a series of lipid-soluble (trans-(R,R)- and (S,S)-1,2-diam inocyclohexane)
platinum(II) complexes incorporated in liposomes, J
Med Chem 1991; 34 (1): 325.
3. Khokhar AR, Al-Baker S, Perez-Soler R. Toxicity and
efficacy studies on a series of lipid-soluble dineodecanoato(trans-R,R - and trans-S,S-1,2-d iaminocyclohexane)platinum (II) complexes entrapped in liposomes.
Anticancer Drugs 1992; 3: 95.

4. Perez-Soler R, Khokhar AR, Lopez-Bereste in G. Treatment and prophylaxis of experimental liver metastases
of M5076 reticulosarcoma with cis-bis-neodecano atotrans-R,R-1,2-d iaminocyclohexa neplatinum (II) encapsulated in multilamellar vesicles, Cancer Res 1987; 47:
6462.
5. Perez-Soler R, Lopez-B erestein G, Lauterszt ain J, et
al. Phase I clinical and pharmaco logical study of liposome entrapped cis-bis-ne odecanoat o-trans-R ,R1,2-diamm inocycloh exaneplat inum (II). Cancer Res
1990; 50: 4254.
6. Perez-Soler R, Han I, Al-Baker S, Khokhar AR.
Lipophilic platinum complexes entrapped in liposomes:
improved stability and preserved antitumor activity with
complexes containing linear alkyl carboxylato leaving
groups. Cancer Chemother Pharmacol 1994; 33: 378.
7. Perez-Soler R, Shin DM, Siddik ZH, Murphy WK, Huber M, Lee JS, Khokhar AR, Hong WK. Phase I clinical and pharmacological study of liposome-entrap ped
NDDP administered intrapleurally in patients with malignant pleural effusions, Clin Cancer Res 1997; 3: 373.
8. Lvi F. Chronopharma cologie et chronoth rapie des
cancers. Pathologie Biologie 1996; 44 (7): 631.
9. Lvi F. Chromotherapy for gastrointestinal cancers.
Curr Opin Oncol 1996; 8: 334.
10. Souli P, Raymond E, Brienza S, Cvitkovic E, Oxaliplatine: le premier DACH platine en clinique. Bull Cancer 1997; 84 (6): 665.
11. Perez-Soler R, Yang LY, Drewinko B, Lautersztain J,
Khokhar AR. Increased cytotoxicity and reversal of resistance to cis-diammined ichloroplatinum (II) with entrapment of cis-bis-neodecanoa to-trans-R,R-1 ,2-diamminocyclohexane platinum (II) in multilam ellar lipid
vessels. Cancer Res 1988; 48: 4509.
12. Perez-Soler R, Khokhar AR. Lipophilic cisplatin analogues entrapped in liposomes: role of intraliposomal
drug activation in biological activity. Cancer Res 1992;
52: 6341.
13. Seiler G, Sigel A, Sigel H (eds) Handbook on Metals
in Clinical and Analytical Chemistry, New York, Marcel Dekker, Inc., 1994.
14. Akaboshi M, Kawai K, Maki H, Akuta K, Ujeno Y, Ono
K, Miyahara T. Determination of the target volume of
HeLa cells treated with platinum -195m radiolabeled
trans-diaminodic hloroplatinum (II): a comparison with
cis-diaminodichl oroplatinum (II). Nucl Med Biol 1993;
20 (4): 389.

15. Hoeschel e JD, Butler TA, Roberts JA. Correlati on of

physico-ch emical and biologica l properties with vivo
biodistrib ution data for platinum -195m-la beled
chloroam mineplatinu m(II) complexe s. Inorg Chem
Biol Med 1980; 140: 181.
16. DeSimone PA, Yancey RS, Coupal JJ, Butts JD,
Hoeschel JD. Effect of a forced diuresis on the distribution and excretion (via urine and bile) of 195m platinum when given as 195m platinum cis-dichlorodiamineplatinum (II). Cancer Treatment Reports 1979; 63
(6): 951.
17. Bnard P, Desplanches G, Macquet JP, Simon J. Wholebody autoradiographic study of the distribution of
195m-Pt in healthy and tumor-bearing mice treated with
labeled cisplatin. Cancer Treatment Reports 1983; 67
(5): 457.
18. Robins AB, Leach MO. Pharm acokinetics of therapeutic doses of isotopically labeled platinum antitumor
agents in the mouse and rat. Cancer Treatment Reports
1983; 67 (3):245.
19. Khokhar AR, Al-Baker S, Krakoff IH, Perez-Soler R.
Toxicity and antitumor activity of cis-bis-carboxylato(trans-R,R- 1,2-diaminocyclo hexane)platinum (II)
Complexes entrapped in liposomes. Cancer Chemother
Pharmacol 1989; 23: 219.
20. Perez-Soler R, Francis K, Al-Baker S, Pilkiewicz F,
Khokhar AR. Preparation and characterization of liposomes containing a lipophilic cisplatin derivative for
clinical use. J Microencapsul 1994; 11 (1): 41.
21. Chaney SG, Wyrick SD, Till GK. In vitro biotransformations of tetrachloro(d,l-tra ns)-1,2-diaminoc yclohexaneplatinum( IV) (tetraplatinum) in rat plasma. Cancer Res 1990; 50: 4539.
22. Screnci D, Er HM, Hambley TW, Galettis P, Brouwer
W, McKeage MJ. Stereoselective peripheral sensory
neurotoxicity of diaminocyclohe xane platinum enantiomers related to orm aplatin and oxaliplatin. Br J Cancer 1997; 76 (4): 502.
23. Mauldin SK, Gibbons G, Wyrick SD, Chaney SG. Intracellular biotransformati on of platinum compounds
with the 1,2-diaminocyc lohexane carrier ligand in the
L1210 cell line. Cancer Res 1988; 48: 5136.
24. Mauldin SK, Plescia M, Richard FA, Wyrick SD, Voyksner RD, Chaney SG. Displacement of the bidentate malonate
ligand
from
(d,l-1,2-diaminocyclohexane)
malonateplatinum(II) by physiologically important compounds in vitro. Biochem Pharmacol 1988; 37 (17): 3321.

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