Potential Future Application With Therapeutic Agents H a r o l d L. A t k i n s
Several n e w approaches to radiation therapy with radionuclides have been discussed. Iron 55 is selectively utilized in the red cell developmental cycle and in therapeutic doses, can lower m a r r o w and circulating erythrocyte levels with much smaller degrees of effect on other cell lines. A serious complication, noted in animal studies, is the induction of neoplasma, especially osteosarcoma. Selective irradiation of the cell nucleus is possible with lzSlUdR. This results in highly efficient cell killing due to the highly
concentrated region of ionization. High concentrations of densely ionizing radiation in the malignant cell m a y also be accomplished with 211At. The use of labeled liposomes is an additional approach to the delivery of intracellular irradiation. None of these approaches is applicable for the practical treatment of human malignancy at the present time. The importance of these approaches is their value as models for future development of methods that can provide highly selective radiation to target sites.
RAPID G R O W T H in nuclear medicine T HEduring the past two decades has resulted in
EFFECT OF IRON 55 ON RED CELL PRODUCTION
a proliferation of the variety and number of diagnostic procedures performed. A similar rate of development has not been apparent in the therapeutic application of radionuclides. In fact, the number of therapeutic procedures with radionuclides has probably declined as chemotherapy has improved. The initial promise of the "magic bullet" homing in on the target has evolved only in the use of radioactive iodine for thyroid disease. Phosphorus-32 is somewhat less specific in its effects on hematologic conditions and other malignancies. The use of radioactive colloids has in large part given way to chemotheropeutic agents. The list of topics included in this issue of Seminars is indicative of the rather limited clinical application of radionuclides in therapy prevalent at this time. The recent convening of a conferer~ce on radiation therapy with radionuclides may signal a reversal of the trend. ~ Advances in biochemistry, radionuclide production, immunology, and knowledge of cell kinetics are being applied to the study of a number of possibilities for therapy of malignant conditions with radioactive materials. Much of the emphasis of recent research is on subcellular localization and the consequent effects of localization of highly ionizing radiation with very short ranges in tissue. The transport of these nuclides to target sites depends on specific metabolic routes, incorporation into appropriate compounds, or protection from diversion into other metabolic processes. High specificity may be achieved through the use of immune mechanisms or binding to receptor sites.
High selectivity of radiation effect on red cell precursors can be achieved by incorporated radioiron. Radiation effects with iron 55 can be confined primarily to cells in the erythropoietic series] This has been demonstrated in studies performed on mice, but has not been applied to human disease. The decay characteristics of 2.7-yr iron 55 are given in Table 1. tt can be seen that the major components are Auger electrons of very low energy, with a range of less than 1 #m in water. Thus, the radiation is largely confined to the erythroblast or erythrocyte. The confinement of radiation damage to the erythropoietic series has been demonstrated in mice after administration of 0.7-15 mCi per animal. ~ A severe diminishing of marrow erythroblasts to 10% of control levels was noted in addition t o depression of tracer ~gFe uptake in peripheral blood (Figs. 1,2). Depression of marrow and peripheral cells in the lymphoid and granulopoietic series was much less. There was a loss of stem cells, as demonstrated by the decreased ability of transplanted marrow to prevent irradiation death. This decrease in hemopoietic stem cells in 55Fe-
Seminars in Nuclear Medicine, Vol. IX, No, 2 (April), 1979
From the Medical Department, Brookhaven National Laboratory, Upton, N. K, and the Department of Radiology, Health Sciences Center, State University o f New York, Stony Brook, N. Ii. Reprint requests should be addressed to." Harold L. Atkins, M.D., Department o f Radiology, Health Sciences Center, State University o f New York, Stony Brook, N. 1L 11794. 9 1979 by Grune & Stratton, Inc. O001 2998/79/0902-0007502.00/0
121
122
HAROLD L. ATKINS
Table 1. E n e r g y D i s t r i b u t i o n o f Iron 55 Decay 2 Frequency/ Disintegration Auger electrons
5.33
K-Shell x-rays
0.2345
Mean Energy (eV)
90%
50%
889
7 . 5 8 X 10 - H
< 1 #m
--
4,2%
5,961
2 . 2 3 6 X 10 -11
980 #m
290/~m
EFFECT OF 55-1RON ON ABSOLUTE CELL COUNTS
extremely high specific activity in order not to exceed the iron-binding capacity of blood) At this time it is difficult and expensive to obtain 55Fe of the requisite specific activity. More disturbing is the induction of neoplasms in irradiated mice. 6 In 14 animals receiving 0.7 or 1.4 mCi and surviving more than 300 days, there were 12 neoplasms. Among these, 6 were osteosarcomas and the other 6 were hematopoietic neoplasms, including leukemia, hemangioendothelioma, and thymic lymphoma. Only one tumor, a reticulum cell neoplasm, was found in 24 control mice. Radioactive, iron is known to accumulate in bone] In the previously mentioned study, 6 autoradiographs demonstrated 5SFe in macrophages, reticulum cells, sinus endothelium, and in endosteal and periosteal cells. The distribution may account for the observed incidence of tumors. BIOLOGIC EFFECTS OF INTRANUCLEAR 1251
Iodine 125 also decays by electron capture with the emission of large numbers of Auger PER HIND LEG
9
20
NUCLEATEDRED CELL SERIES q
Range in Water
95,8%
treated mice is probably not directly due to radiation injury, and may be secondary to an increased turnover as a response to the increased destruction of maturing erythroblasts) "4 Continuing cytocide of cells in the erythrocyte series occurs through reutilization of the 55Fe released from disintegrating cells. Calculations of absorbed dose by Reincke et al. 2 indicate that an erythroblast of 300 cu #m containing 55Fe as 5% of its final iron content may receive a dose of 250 rad/hr, which will increase to t 300 rad/hr at a mature red cell size of 60 cu #m. Such a cell would not survive to reach the peripheral blood. Radiation levels to adjacent cells would be lower by more than ten orders of magnitude. The low radiation effect on cells of extracellular 55Fe was experimentally demonstrated, inasmuch as bone marrow cells exposed to extracellular iron 55 showed no loss of colony-forming ability) There are two factors that presently inhibit the application of 55Fe cytocide to the treatment of polycythemia. Iron 55 must be obtained in
I0
Total Energy (g. rod/disintegration)
I.
e
GRANULOCYTIC SERIES 9 LYMPHOID CELLS * OTHER CELLS 9
6-
16 12
z 4-
§ I
LLJ
o
oo
SL
,, $
lO l'2 14 16 0
0
55-iRON DOSE, mCi mCi 55Fe dayspostini. Controls days post ini. 0.13 0.6 1.3 2 2 9.4 15
5 5 5
2 5 5 7
Cold Fe Cold Fe untreated
+
2 7
Fig. 1, Effect of iron 5 5 o n marrow precursor cells is shown with increasing dose of iron 55. There is a marked decrease in numbers of cells in the erythropoietic series in the hind leg of treated mice. Lesser effects are noted on cells in the granulocytic, lymphoid, and " o t h e r " series. Doses and killing intervals noted in table (reproduced with permission=).
123
POTENTIAL FUTURE APPLICATIONS
59Fe-TRACERUPTAKEIN BLOODOF 55Fe-TREATEDMICE 59Fe6 HRSAFTER55Fe 59Fe70 HRSAFTER55Fe
59Fe12 DAYS
Table 2. Calculated Gamma-, X-ray, and Electron Radiation of lZSla
6O o,
5O
~///~////////////////.~ ~,}'/////z///////////~
COLD-IRON-GONTROLS
Gamma Radiation
J-I
n/lO0 disintegration
Electron Radiation
n/lO0 disintegra~ tion
m ,,=, 4O
a_
3.7 keY (X-ray-LM)
i)
27,5 keV
3O
(X-ray-K(x 0
i ~- 2O
(X-ray-K(z 2)
I 2.8
I I I I I 0.7 IA 2.8 0.7 1,4 INJECTEDDOSE mCi
80
74.3
(L-conversion)
11
34.6 keV 37.9
(M-conversion)
2
31.0 keV
,w I I 0.7 1.4
3.6 keV (K-conversion) 31.0 keV
27,2 keV
,i
I0 0
22.0
(x-ray-K#) I 2.8
Fig. 2. Suppression of iron 59 tracer in the cells in peripheral blood at 6 hr, 7 0 hr, and 12 days after iron 55 t r e a t m e n t in mice (reproduced with permission=).
electrons per disintegration s (Table 2). It has been investigated and used as a possible therapeutic agent in hyperthyroidism. The concentration of the radioactive iodine in the colloid follicle and the limited range of the low-energy Auger electrons was thought to be advantageous in the prevention of late hypothyroidism by sparing the follicular cell nucleus from radiation effects. 9 Several therapeutic trials have been instituted, but the results have not been especially gratifying in most cases, m ~z In an opposite approach, the biologic effects of intranuclear deposition of iodine 125 are being investigated as a more effective method for tumor therapy. Studies have been carried out to establish the reasons for the marked biologic effectiveness of iodine 125 when incorporated into the cell nucleus, compared to other radionuclides having the same biologic distribution. Hofer and Hughes demonstrated an enhanced radiation effect of incorporated ~25I-iododeoxyuridine on mouse L1210 lymphoid leukemia cells grown in the peritoneal cavity, in comparison with iododeoxyuridine '3~1 and tritiated thymidine. 14 The effects of ~3~I-IUdR and 3H-TdR were very similar, whereas '25I-IUdR demonstrated greater cell killing by a factor of 4-5 (Fig. 3). The reduction of surviving fraction of the cells to 50% required 4 disintegrations/cellhr for ~2sI, whereas ~3~I required 47, and 3H required 62 disintegrations/cell-hr for the same effect. It was calculated that the energy deposited in the nucleus was 300 keV/hr for 3H, 60 keV/hr for 125I, and ~ 1 2 0 keV/hr for 131l.
20.1
22.7 keY (KLL)
17
31.7 keV (x-ray-K v) 35.4 keV (unconverted)
4.5 7,0
26.3 keV (KLM)
7
3.0 keV (LMM) 3,5 keV (LMN) 0,5 keV (MNN)
107 54 265
0.5 keV (MNO)
34
The increased efficacy of 12-sl when incorporated into DNA has been attributed to the severely constricted distribution of the energy of decay resulting from multiple electrons, with a high possibility of damage to both strands of the DNA. ~5't6 Seventy percent of the electrons have initial energies of less than 0.5 keV and produce, on the average, one ionization per 20 ~ in unit density material. Another possible explanation for the greater effect of '25I-IUdR could be disruption of the molecule during the decay process, which produces a heavily positive-charged tellurium atom) v This is a highly unstable situation, in which electrons are attracted from other atoms in the molecule, as demonstrated by Carlson and White L~and Wexler ~9in organic molecules in the gaseous phase. Hofer, Keough, and Smith investigated this possibility and showed, by a double-labeling technique, that molecular fragmentation is probably not a factor in a cellular system. ~5 Column chromatography was carried out on a sample of 14C-t2~IUdR incubated for 76 days at 4 ~ C. The principal effect was noted to be deiodination, with all the t4C contained in a single peak of the eluted material. Furthermore, it was estimated that deiodination occurred with virtually 100% efficiency. The increased biologic effect of ,25| is entirely a consequence of its intranuclear localization when labeled to deoxyuridine. The intense,
124
HAROLD L. ATKINS
I00
[I
~ =
ii
90- 1 8O70-
6050Z o
I--
40-
30N N
aH
m
2013i I
1261
|@ - - 0
I I0
[---20
T
50
I
40
J
50
l
60
d/cell/hr
extremely localized ionization events must involve the genetic apparatus in the cell nucleus. Iodine 125 irradiation of the cell membrane and cytoplasm (by labeled concanavallin A, Fig. 4) is no more effective than other forms of radiation in causing loss of reproductive integrity. 2~ This theory is strengthened by studies on V79 Chinese hamster cells in vitro. The efficacy of cell killing corresponded to ability to induce chromosome breaks in a comparison of ~25IUdR, 13tIUdR, and 3HTdR 2~ (Fig. 5). Application of ~2SlUdR to clinical cancer therapy is not a trivial problem. Intravenous administration does not lead to selective localization, and rapid deiodination occurs. Uptake in cells occurs only during the DNA synthesis phase of cell growth. Nevertheless, the high efficiency of I25IUdR in producing cell death has stimulated some investigation into its possible use in cancer therapy. An ascites tumor model in the C3HeB/FeJ mouse has been investigated] 2-25 Intraperitoneal
~
70
t
80
Fig. 3. Fraction of L1210 cells surviving various radioisotope doses, plotted as a function of disintegrations/cell-hr. The lack of a shoulder to the 1251 curve is characteristic of high LET-type radiation (reproduced with permission14).
administration of the 125IUdR permits direct access to the tumor cells. Fractionated therapy was used in order to expose all cells during the DNA synthesis phase. As in other systems studied, an increased efficacy of ~25IUdR as compared to ~3~IUdR was demonstrated (Fig. 6). Application to human cancers is not immediately obvious. Possibilities are suggested, however. Perhaps direct intraarterial administration to tumor sites might be efficacious when dehalogenation beyond the tumor protects the rest of the body. Other agents with high nuclear affinity should be sought. Given enough concentration of a high capture cross-section nuclide, the possibility for neutron capture therapy exists. SELECTIVE IRRADIATION WITH ASTATI N E-21 1
There has been a recent renewal of interest in the chemistry of astatine, with the hope of incorporating astatine 211 into organic molecules in
POTENTIAL FUTURE APPLICATIONS
125
/251UdR
6 4
'-,~
i
2 0
"M
L ~ ___
'_L~_
'
_~___
I
/s/IUdR
% \
i~~~brone 125I
oz~u 0 Hours 9 9 Hours
4 12
~
-
A
~
z~
JllrdR
6
D
E3
E3
9
E3 r
,,\
~
0
9
0.5
f.O
Dc~ ia
J f.5
I 20
j
2.5
pC~/CELL ,
I
800 DOSE (rod)
i
I
1200
Fig. 4. Fraction of Chinese hamster ovary cells surviving various doses of 12SI-UdR (nucleus), 3H-TdR, or concanavallin A 1251(membrane), plotted as a function of radiation dose to the cell nucleus (reproduced with permission2~
order to accomplish selective radiation of specific cellular types or organs. Decay of 7.2-hr 2~At occurs with emission of a 5.87-MeV alpha particle 42% of the time. The other 58% of decays are by electron capture to 0.5-sec 2~Po, which in turn emits 7.45-MeV alpha particles. 26 Astatine belongs to the halogen group, with some similarities to iodine. The biologic distribution of this element was studied almost immediately after its first production in 1940. 27 Uptake in the thyroid was lower than for radioiodine, but selective localization in the thyroid was definitely apparent. The destructive action of the densely ionizing, high-LET alpha particles was demonstrated in histologic examination of rats that had received various amounts of 2~AT.2~ Observations were also extended to patients receiving 2~JAt preoperatively by oral administration. 29 Thyroidal uptake was as great as 17%, and the degree of uptake appeared to correlate with iodine content. Prior administration of
Fig. 5. Total chromosome breaks (chromatid and chromosome type) induced in V79 Chinese hamster cells immediately (0 hr) or 24 hr after removal of lZSIUdR, lzllUdR, or 3H-TdR. The lines are drawn by eye-fit (reproduced with permission 21), 1311UdR
,-~i i~ 10-2 2 ~ 10_3.
~2sIUdR
io-4 i
10-5[
0
Fig. 6.
L gO
L I 1 I 40 60 80 I00 /z,' d'//DOSE, q 4 h x 4
Dose-response curve for 12SlUdR and ?3~IUdR
therapy in a mouse ascites tumor. Animals were treated every 4 hr for four doses, starting 24 hr after the i.p. injection of 10 s tumor cells (reproduced with permission23).
126
HAROLD L. ATKINS
stable iodine decreased uptake. Concentration in papillary adenocarcinoma contained in a lymph node was very low. While accumulation of astatine may be increased in the thyroid by prior administration of agents that block organification of iodine, the application of 2'~At to therapy of thyrotoxicosis or thyroid malignancy has not been accomplished. It seems unlikely that radioastatine will be used for this purpose. Of more recent interest has been the possibility of labeling various organic molecules with 2~At for the purpose of selective destruction of specific cells, while sparing nearby cellular elements that do not accumulate the compound. Hughes and Gitlin 3~ as far back as 1955 attempted the labeling of proteins with 21~At, but the products were not stable. The usual methods utilized in radioiodine labeling have not yielded good results with astatine. Electrooxidation and the use of hydrogen peroxide in the presence of 0.2 #M KI have produced yields of 30%-60% in labeling of proteins. 3~ Zalutsky et al. 32 have accomplished stable binding of astatine to bovine serum albumin, with 12% yield. The labeled material retained its immunospecific properties. Labeling was carried out in a two-step process, first forming an astatinated benzoic acid and then conjugating this to the serum albumin. Another method of labeling with 2~At has been via the decomposition of 5-diazonium salts in the presence of ionic astatine to form 5astatouracil and 5-astatodeoxyuridine. 33'34 Yields of 30% have been obtained. Animal distribution studies in normal and tumor mice have been carried out. 34 The astatinated compounds appear to have a greater concentration in tumor tissue than the corresponding iodinated compound by a factor of 3.
A
B
~lprn
Further work is being carried out to establish the biologic effects and distribution of astatinelabeled compounds. Possibilities include tumorseeking materials, labeling of immunospecific proteins, and destruction of lymphocytes for prevention of transplant rejection. 35'36 LIPOSOMES AS CARRIERS OF THERAPEUTIC AGENTS
Research into the properties of lipid bilayers has resulted in the continuing development, during the past decade, of multilamellar vesicles or liposomes as carriers to deliver biologically active materials into cells. Liposomes are formed by evaporating to dryness a solution of lipid in an organic solvent and then dispersing the resulting film of lipid in water or aqueous buffer. 37'38 Multilamellar concentric bilayer vesicles are formed, consisting of alternating layers of lipid and aqueous medium. Sonication may be used to decrease the size of liposomes. Size may range from 25 nm to 1 #m (Fig. 7). Various materials, such as large proteins, enzymes, radiopharmaceuticals, and chemotherapeutic agents, can be entrapped in liposomes. The degree of entrapment is low, and separation of the liposomes from the free solution is necessary. This may be accomplished by gel filtration, centrifugation, and dialysis. The possibility of using liposomes to deliver therapeutic materials to cells has a number of advantages. 39 First, the biologically active material is protected from degradation in the blood. In addition, the liposomes are able to carry the material across the cell membrane and into the cytoplasm, where the subsequent release of the entrapped material can affect cell metabolism. By the use of various methods of targeting the liposomes, the active material can be directed to specific locations, thus sparing other tissues from
Lipid Biloyer
tarnellae
C
,~_..~
Aqueous Spaces
_ ~ 25 nm
Fig, 7. Schematic representations of liposomes. (a) Multilamellar liposome. (b) Enlarged view of (a). (c) Small unilamellar liposome (reproduced with permission37).
POTENTIAL FUTURE APPLICATIONS
127
the biologic effects. Delayed elimination and decreased chances for allergic reactions are characteristic of liposome-entrapped drugs. The distribution of liposomes administered i.v. is primarily to the reticuloendothelial system. Incorporation into cells may be by endocytosis or by fusion with the cellular plasma membrane4~ (Fig. 8). Distribution may be affected by liposome size, charge, or incorporation of immunospecific globulins. Disappearance from the blood is fairly rapid during the first 2 hr, but a significant proportion of the injected dose may still be circulating several hours after administration (Fig. 9). Slower clearance is evident for the smaller liposomes. Much of the interest in liposomes is related to the delivery of chemotherapeutic agents in the treatment of malignancy and enzymes in deficiency and storage disorders. Research into the application of this system to the use of radionuclide therapy is limited. Radioactivity may be added to liposomes by including the labeled compound in the aqueous medium that is used to prepare the liposomes. Labeling may also be accomplished after formulation of the liposomes. For example, entrapped albumin has been labeled with radioactive iodine using the monochloride method4~ and bleomycin has been labeled with radioactive indium.4] Nucleus
i~
kysosome /
mamembrane
~. Drug-containing liposome Fig. 8. Cellular uptake and lyzosomotropic action of liposome-entrapped drugs. The liposome is taken up by endocytosis. The endocytic vacuole fuses with a lyzosome, the hydrolases of which disrupt the lipid bilayers of the liposome, releasing the entrapped drug. The drug is then able to diffuse out and act in other cellular compartments (reproduced with permission4~
40, 30q
20 x
~8
; ~.
8
7 5 84
2 9
1
2
3
&
4
5
6
Daysafter Injection Fig. 9. Trichloroacetic acid-precipitable radioactivity in plasma of three patients injected with liposome-entrapped lZ'l-labeled albumin (reproduced with permission4~
Labeled antibodies and other proteins may be attached to the outer membrane. 42 Liposomes were labeled with 99mTcusing a SnCI 2 reduction method, by Richardson et al., with 97% efficiency.43-45The SnCI 2 was added to the preformed liposomes, followed by a pertechnetate solution. Distribution studies in rats were performed by imaging of and killing of the animals after administration of positive, negative, and neutral liposomes. Negatively charged liposomes had the slowest blood clearance, followed by positively charged and neutral ]iposomes. Distribution in organs at 29 hr was similar for positive and neutral liposomes, with the major portion of the administered radioactivity in liver (~24%), spleen (~17%), stomach and intestines (~7-8%), and kidneys (~9%). Negatively charged liposomes demonstrated somewhat less liver uptake (11.88%) and more kidney uptake (11.06%). Tumor-bearing (Walker 256 carcinoma) rats were also studied, and demonstrated a similar pattern of distribution. It was possible to image tumors at 12 hr with positive and negative liposomes (Fig. 10). Tumor uptake of negatively charged liposomes could be increased by a factor of 7 with extensive sonication. Maximum tumor
128
HAROLD L. ATKINS
TUMOUR
C
B
TUMOUR
TUHOUR
Fig. 10. Computer-drawn contour mapping of 99mTc in rats bearing Walker 256 carcinoma. (a) S9mTcStannous colloid. (b) Anionic liposomes. (c) Neutral liposomes. (d) Cationic liposomes. The tumor is best outlined with anionic liposomes labeled with 99"Tc (reproduced with permission4S).
concentration was 1.3%/g compared to liver concentration of 1.62%/g, spleen concentration of 3.96%/g, and kidney concentration of 8.72%/8. The same investigators also studied twelve patients with an assortment of malignant tumors. 44 In none was significant localization in tumor apparent by imaging procedures. Liver and spleen uptake were readily noted, but no uptake was observed in two patients with hepatoma. In some patients there was marked uptake in the bone marrow. This was especially apparent in one patient with polycythemia vera, in whom there was very little liver uptake. In another study involving human patients, Gregoriadis et al? ~ were able to obtain tissue samples in two of three patients administered t3q-human serum albumin-containing liposomes. Examination of the tissues at autopsy 5 days after administration of the liposomes was carried out in a patient with transitional cell carcinoma of the bladder, with liver and spleen metastasis. In this patient, 81% of the radioactivity injected was found in the liver. The primary tumor in the kidney had 50 times the concentration of radioactivity of normal kidney, but the differential between normal liver and liver metastasis was negligible. Of interest was evidence of lack of catabolism of the labeled albumin in the primary tumor. This was not true for metastatic deposits or normal tissue. The other patient had a resection of a colon
tumor that had metastasized to the liver. Tissue samples were obtained 3 hr after injection. Again, a negligible difference was found in liver metastasis and normal liver, while the colon tumor had slightly more than two times the concentration of radioactivity of normal colon. Lymph node uptake of neutral or positively charged liposomes has been noted after interstitial administration of liposomes labeled with 99mTC.44
To date, the major interest and possibilities of liposomes are related to the delivery of biologically active compounds to cells. 46 52 The use of radioactive materials has been extremely limited, and there would seem to be little advantage to the use of radiation over chemotherapeutic modalities. Much investigation is needed to improve the specific targeting of liposomes. An example is the attachment of immunospecific molecules on the liposome surface. 42 When this is achieved, it may be possible to incorporate alpha-emitting or Auger electron-emitting radionuclides in order to provide high, LET-type radiation limited to specific cell types. An additional possibility is the e n t r a p m e n t of highcapture, cross-section nuclides, which could be delivered to tumors or organs for exposure to a neutron beam for localized radiation effects. ACKNOWLEDGMENT
The assistance of Drs. Ursula Reincke, Medical Department, Brookhaven National Laboratory, and Clifton R. Harris, Harvard Medical School, is greatly appreciated.
POTENTIAL FUTURE APPLICATIONS
129
REFERENCES
1. RP Spencer (ed): Therapy in Nuclear Medicine. New York, Grune & Stratton, 1978 2. Reincke U, Burlington H, Cronkite EP, et al: Selective damage to erythroblasts by ~Fe. Blood 45:801 810, 1975 3. Reincke U, Burlington It, Cronkite EP, et al: Hayflick's hypothesis: an approach to in vivo testing. Fed Proc34:71 75, 1975 4. Reincke U, Brookoff D, Burlington H, et al: Susceptibility of hematopoietic stem cells (CFU-s) to 55Fe radiation damage. Radiat Res 74:66 78, 1978 5. Reincke U, Brookoff D, Cronkite EP, ct al: Relevance of specific activity in experimental crythrocytocidc by 55Fc. Experientia (in press) 6. Laissue JA, Burlington H, Cronkite EP, et al: Induction of osteosarcomas and hemalopoietic neoplasms by ~SFe in mice. Cancer Res 37:3545 3550, 1977 7. Thomson RAE, Corriveau O J, Rubin P: 59Fc labelling inbone. J N u c l M e d 15:161 163, 1974 8. Ertl HH, Feinendegcn LE, tteiniger H J: Iodine-125, a tracer in cell biology: physical properties and biological aspects. Phys Med Biol 15:447 456, 1970 9. Lewitus Z, Lubin E, Rechnic J, et al: Treatment of thyrotoxicosiswith~251and~l. SeminNuclMed 1:411 421, 1971 10. Siemsen J, Wallack MS, Martin RB, et al: Early results of ~251therapy of thyrotoxic Graves" disease. J Nucl Med 15:257 260, 1974 11. Weidingcr P, Johnson PM, Werner SC: Five years" experience with iodine-125 therapy of Graves's disease. Lancet 2:74-77, 1974 12. Glanzmann C, Horst W: Ergcbnissc dcr Therapie der Hyperthyreoscmit 125-Jod. NuclMcd(Stuttg) 14:207 218, 1975 13. Gimlctte TMD, Hosehl R: Treatment of thyrotoxicosis with iodine-125 in moderately low dosage. Clin Radiol 24:263 266, 1973 14. Hofer KG, Hughes WL: Radiotoxicity of intranuelear tritium, ~"Siodineand ~3~iodine. Radiat Res 47:94 109, 1971 15. Hofer KG, Keough G, Smith JM: Biological toxicity of Auger emitters: molecular fragmentation versus electron irradiation. CurrTopRadiat Res. 12:335 354, 1977 16. Feinendegen LE: Biological damage from the Auger effect, possible benefits. Radiat Environ Biophys 12:85 99, 1975 17. Carlson TA, White R M: "Explosion" of multicharged molecular ions: chemical consequences of inner shell wlcancies in atoms, in: Chemical Effects Associated With Nuclear Reactions and Radioactive Transformations. Proceedings of a Conference, vol I. Vienna, IAEA. 1965, pp 23 33 18. Carlson TA, White RM: Formation of fragment ions from Ctt~Te ~25 and C2H~Tc125 following the nuclear decays ofCH~l ~'~ and C2H~112~. J Chem Phys 38:2930 2934, 1963 19. Wexler S: Destruction of molecules by nuclear transformations. Science 156:901 907, 1967 20. Warters RL, Hofcr KG, Harris CR, et al: Radionuelide toxicity in cultured mammalian ceils: elucidation of the primary site of radiation damage. Curr Top Radiat Res 12:389 407, 1977 21. Chan PC, Lisco E, lfisco H, ctal: The radiotoxicity of iodine-125 in mammalian cells. 11. A comparative study on
cell survival and cytogenetic responses to ~251UdR, ~3qUdR, and 3HTdR. Radiat Res 67:332-343, 1976 22. Bloomer WD, Adelstein S J: Therapeutic application of iodine-125 labelled iododeoxyuridine in an early ascites tumor modeI. CurrTopRadiatRes12:513 525, 1977 23. Bloomer WD, Adelstein S J: 5-(t2~l)-iododeoxyuridine in experimental tumor therapy, in Spencer RP (ed): Therapy in Nuclear Medicine. New York, Grune & Stratton, 1978, pp 177-183 24. Bloomer WD, Adelstein S J: Antineoplastic effect of iodine-125-1abelled iododeoxyuridine. Int J Radiat Biol 27:509-511, 1975 25. Bloomer WD, Adelstein S J: 5J251-iododeoxyuridine as a prototype for radionuclide therapy with Auger emitters. Nature 265:620-621, 1977 26. Lederer CM, Hollander JM, Perlman 1: Table of Isotopes (ed 6). New York, Wiley, 1967 27. Hamilton JG, Soley MH: A comparison of the metabolism of iodine and of element 85 (eka-iodine). Proc Natl AcadSciUSA26:483 489, 1940 28. Hamilton JG, Asling CW, Garrison WM, et al: Destructive action of astatine TM (element 85) on the thyroid gland ofthe rat. ProcSocExpBiolMed73:51 53, 1950 29. Hamilton JG, Durbin PW, Parrott MW: Accumulation of astatine 2~ by thyroid gland in man. Proc Soc Exp Biol Med 86:366 369, 1954 30. Hughes WL, Gitlin D: Astatinated (At 2~) organic compounds for production of highly localized radiobiological effects. Fed Proc 14:229, 1955 31. Aaij C, Tschroots WRJM, Lindner L, et al: The preparation of astatine labelled proteins, lnt J Appl Radiat Isot26:25 30, 1975 32. Zalutsky MR, Friedman AM, Buckingham FC, et al: Synthesis of a non-labile astatine-protein complex. J LabelledCompRadiopharm 13:181 182,1977 33. Meyer G J, R6ssler K, St6cklin G: Preparation and high pressure liquid chromatography of 5-astatouracil. J Labelled Comp Radiopharm 12:449-458, I976 34. R6ssler K, Meyer G3, St6cklin G: Labelling and animal distribution studies of 5-astatouracil and 5-astatodeoxyuridine (2~At)..I Labelled Comp Radiopharm 13:27 I, 1977 35. Myburgh JA, Smit JA: Enhancement and antigen suicide in the outbred primate. Transplant Proc 5:597-600, 1973 36. Smit JA, Myburgla JA, Neirinckx RD: Specific inactivation of sensitized lymphocytes in vitro using antigens labelled with astatine-211. Clin Exp lmmunol 14:107 116, 1973 37. Tyrrell DA, Heath TD, Colley CM, et al: New aspects of liposomes. Biochim Biophys Acta 457:259 302, 1976 38. Poste G, Papahadjopoulos D, Vail W J: Lipid vesicles as carriers for introducing biologically active materials into cells. Methods Cell Biol 14:33 71, 1976 39. Gregoriadis G: Targeting of drugs. Nature 265:407 41 I, 1977 40. Gregoriadis G, Swain CP, Wills EJ, et al: Drugcarrier potential of liposomes in cancer chemotherapy. Lancet 1:1313 1316, 1974
130
41. Neerunjun ED, Hunt R, Gregoriadis G: Fate of a liposome-associated agent injected into normal and tumourbearing rodents: attempts to improve localization in tumour tissues. Biochem Soc Trans 5:1380-1383, 1977 42. Gregoriadis G, Neerunjun ED: Homing of liposomes to target cells. Biochem Biophys Res Commun 65:537-544, 1975 43. Richardson V J, Jeyasingh K, Jewkes RF, et al: Properties of (99mTC)technetium-labelled liposomes in normal and tumour-bearing rats. Biochem Soc Trans 5:290-291, 1977 44. Ryman BE, Jewkes RF, Jeyasingh K, et al: Potential applications of liposomes to therapy. Ann NY Acad Sci 308:281-307, 1978 45. Richardson VJ, Jeyasingh K, Jewkes RF, et al: Possible tumor localization of Tc-99m-labelled liposomes: effects of lipid composition, charge, and liposome size. J Nucl Med 19:1049-1054, 1978 46. Chen JS, Del Fa A, DiLuzio A, et al: Liposome-
HAROLD L. ATKINS
induced morphological differentiation of murine neuroblastoma. Nature 263:604-606, 1976 47. Juliano RL: The role of drug delivery systems in cancer chemotherapy. Prog Clin Biol Res 9:21-32, 1976 48. Neerunjun ED, Gregoriadis G: Tumour regression with liposome-entrapped asparaginase: some immunological advantages. Biochim Soc Trans 4:133-134, 1976 49. Colley CM, Ryman BE: The use of a liposomally entrapped enzyme in the treatment of an artifical storage condition. Biochim Biophys Acta 451:417-425, 1976 50. Kimelberg HK: Differential distribution of liposomeentrapped (3H) methotrexate and labelled lipids after intravenous injection in a primate. Biochim Biophys Acta 448:531-550, 1976 51. Fendler JH, Romero A: Liposomes as drug carriers. Life Sci 20:1109-1120, 1977 52. Gregoriadis G: The carrier potential of liposomes in biology and medicine. N Engl J Med 295:765-770, 1976
Several n e w approaches to radiation therapy with radionuclides have been discussed. Iron 55 is selectively utilized in the red cell developmental cycle and in therapeutic doses, can lower m a r r o w and circulating erythrocyte levels with much smaller degrees of effect on other cell lines. A serious complication, noted in animal studies, is the induction of neoplasma, especially osteosarcoma. Selective irradiation of the cell nucleus is possible with lzSlUdR. This results in highly efficient cell killing due to the highly
concentrated region of ionization. High concentrations of densely ionizing radiation in the malignant cell m a y also be accomplished with 211At. The use of labeled liposomes is an additional approach to the delivery of intracellular irradiation. None of these approaches is applicable for the practical treatment of human malignancy at the present time. The importance of these approaches is their value as models for future development of methods that can provide highly selective radiation to target sites.
RAPID G R O W T H in nuclear medicine T HEduring the past two decades has resulted in
EFFECT OF IRON 55 ON RED CELL PRODUCTION
a proliferation of the variety and number of diagnostic procedures performed. A similar rate of development has not been apparent in the therapeutic application of radionuclides. In fact, the number of therapeutic procedures with radionuclides has probably declined as chemotherapy has improved. The initial promise of the "magic bullet" homing in on the target has evolved only in the use of radioactive iodine for thyroid disease. Phosphorus-32 is somewhat less specific in its effects on hematologic conditions and other malignancies. The use of radioactive colloids has in large part given way to chemotheropeutic agents. The list of topics included in this issue of Seminars is indicative of the rather limited clinical application of radionuclides in therapy prevalent at this time. The recent convening of a conferer~ce on radiation therapy with radionuclides may signal a reversal of the trend. ~ Advances in biochemistry, radionuclide production, immunology, and knowledge of cell kinetics are being applied to the study of a number of possibilities for therapy of malignant conditions with radioactive materials. Much of the emphasis of recent research is on subcellular localization and the consequent effects of localization of highly ionizing radiation with very short ranges in tissue. The transport of these nuclides to target sites depends on specific metabolic routes, incorporation into appropriate compounds, or protection from diversion into other metabolic processes. High specificity may be achieved through the use of immune mechanisms or binding to receptor sites.
High selectivity of radiation effect on red cell precursors can be achieved by incorporated radioiron. Radiation effects with iron 55 can be confined primarily to cells in the erythropoietic series] This has been demonstrated in studies performed on mice, but has not been applied to human disease. The decay characteristics of 2.7-yr iron 55 are given in Table 1. tt can be seen that the major components are Auger electrons of very low energy, with a range of less than 1 #m in water. Thus, the radiation is largely confined to the erythroblast or erythrocyte. The confinement of radiation damage to the erythropoietic series has been demonstrated in mice after administration of 0.7-15 mCi per animal. ~ A severe diminishing of marrow erythroblasts to 10% of control levels was noted in addition t o depression of tracer ~gFe uptake in peripheral blood (Figs. 1,2). Depression of marrow and peripheral cells in the lymphoid and granulopoietic series was much less. There was a loss of stem cells, as demonstrated by the decreased ability of transplanted marrow to prevent irradiation death. This decrease in hemopoietic stem cells in 55Fe-
Seminars in Nuclear Medicine, Vol. IX, No, 2 (April), 1979
From the Medical Department, Brookhaven National Laboratory, Upton, N. K, and the Department of Radiology, Health Sciences Center, State University o f New York, Stony Brook, N. Ii. Reprint requests should be addressed to." Harold L. Atkins, M.D., Department o f Radiology, Health Sciences Center, State University o f New York, Stony Brook, N. 1L 11794. 9 1979 by Grune & Stratton, Inc. O001 2998/79/0902-0007502.00/0
121
122
HAROLD L. ATKINS
Table 1. E n e r g y D i s t r i b u t i o n o f Iron 55 Decay 2 Frequency/ Disintegration Auger electrons
5.33
K-Shell x-rays
0.2345
Mean Energy (eV)
90%
50%
889
7 . 5 8 X 10 - H
< 1 #m
--
4,2%
5,961
2 . 2 3 6 X 10 -11
980 #m
290/~m
EFFECT OF 55-1RON ON ABSOLUTE CELL COUNTS
extremely high specific activity in order not to exceed the iron-binding capacity of blood) At this time it is difficult and expensive to obtain 55Fe of the requisite specific activity. More disturbing is the induction of neoplasms in irradiated mice. 6 In 14 animals receiving 0.7 or 1.4 mCi and surviving more than 300 days, there were 12 neoplasms. Among these, 6 were osteosarcomas and the other 6 were hematopoietic neoplasms, including leukemia, hemangioendothelioma, and thymic lymphoma. Only one tumor, a reticulum cell neoplasm, was found in 24 control mice. Radioactive, iron is known to accumulate in bone] In the previously mentioned study, 6 autoradiographs demonstrated 5SFe in macrophages, reticulum cells, sinus endothelium, and in endosteal and periosteal cells. The distribution may account for the observed incidence of tumors. BIOLOGIC EFFECTS OF INTRANUCLEAR 1251
Iodine 125 also decays by electron capture with the emission of large numbers of Auger PER HIND LEG
9
20
NUCLEATEDRED CELL SERIES q
Range in Water
95,8%
treated mice is probably not directly due to radiation injury, and may be secondary to an increased turnover as a response to the increased destruction of maturing erythroblasts) "4 Continuing cytocide of cells in the erythrocyte series occurs through reutilization of the 55Fe released from disintegrating cells. Calculations of absorbed dose by Reincke et al. 2 indicate that an erythroblast of 300 cu #m containing 55Fe as 5% of its final iron content may receive a dose of 250 rad/hr, which will increase to t 300 rad/hr at a mature red cell size of 60 cu #m. Such a cell would not survive to reach the peripheral blood. Radiation levels to adjacent cells would be lower by more than ten orders of magnitude. The low radiation effect on cells of extracellular 55Fe was experimentally demonstrated, inasmuch as bone marrow cells exposed to extracellular iron 55 showed no loss of colony-forming ability) There are two factors that presently inhibit the application of 55Fe cytocide to the treatment of polycythemia. Iron 55 must be obtained in
I0
Total Energy (g. rod/disintegration)
I.
e
GRANULOCYTIC SERIES 9 LYMPHOID CELLS * OTHER CELLS 9
6-
16 12
z 4-
§ I
LLJ
o
oo
SL
,, $
lO l'2 14 16 0
0
55-iRON DOSE, mCi mCi 55Fe dayspostini. Controls days post ini. 0.13 0.6 1.3 2 2 9.4 15
5 5 5
2 5 5 7
Cold Fe Cold Fe untreated
+
2 7
Fig. 1, Effect of iron 5 5 o n marrow precursor cells is shown with increasing dose of iron 55. There is a marked decrease in numbers of cells in the erythropoietic series in the hind leg of treated mice. Lesser effects are noted on cells in the granulocytic, lymphoid, and " o t h e r " series. Doses and killing intervals noted in table (reproduced with permission=).
123
POTENTIAL FUTURE APPLICATIONS
59Fe-TRACERUPTAKEIN BLOODOF 55Fe-TREATEDMICE 59Fe6 HRSAFTER55Fe 59Fe70 HRSAFTER55Fe
59Fe12 DAYS
Table 2. Calculated Gamma-, X-ray, and Electron Radiation of lZSla
6O o,
5O
~///~////////////////.~ ~,}'/////z///////////~
COLD-IRON-GONTROLS
Gamma Radiation
J-I
n/lO0 disintegration
Electron Radiation
n/lO0 disintegra~ tion
m ,,=, 4O
a_
3.7 keY (X-ray-LM)
i)
27,5 keV
3O
(X-ray-K(x 0
i ~- 2O
(X-ray-K(z 2)
I 2.8
I I I I I 0.7 IA 2.8 0.7 1,4 INJECTEDDOSE mCi
80
74.3
(L-conversion)
11
34.6 keV 37.9
(M-conversion)
2
31.0 keV
,w I I 0.7 1.4
3.6 keV (K-conversion) 31.0 keV
27,2 keV
,i
I0 0
22.0
(x-ray-K#) I 2.8
Fig. 2. Suppression of iron 59 tracer in the cells in peripheral blood at 6 hr, 7 0 hr, and 12 days after iron 55 t r e a t m e n t in mice (reproduced with permission=).
electrons per disintegration s (Table 2). It has been investigated and used as a possible therapeutic agent in hyperthyroidism. The concentration of the radioactive iodine in the colloid follicle and the limited range of the low-energy Auger electrons was thought to be advantageous in the prevention of late hypothyroidism by sparing the follicular cell nucleus from radiation effects. 9 Several therapeutic trials have been instituted, but the results have not been especially gratifying in most cases, m ~z In an opposite approach, the biologic effects of intranuclear deposition of iodine 125 are being investigated as a more effective method for tumor therapy. Studies have been carried out to establish the reasons for the marked biologic effectiveness of iodine 125 when incorporated into the cell nucleus, compared to other radionuclides having the same biologic distribution. Hofer and Hughes demonstrated an enhanced radiation effect of incorporated ~25I-iododeoxyuridine on mouse L1210 lymphoid leukemia cells grown in the peritoneal cavity, in comparison with iododeoxyuridine '3~1 and tritiated thymidine. 14 The effects of ~3~I-IUdR and 3H-TdR were very similar, whereas '25I-IUdR demonstrated greater cell killing by a factor of 4-5 (Fig. 3). The reduction of surviving fraction of the cells to 50% required 4 disintegrations/cellhr for ~2sI, whereas ~3~I required 47, and 3H required 62 disintegrations/cell-hr for the same effect. It was calculated that the energy deposited in the nucleus was 300 keV/hr for 3H, 60 keV/hr for 125I, and ~ 1 2 0 keV/hr for 131l.
20.1
22.7 keY (KLL)
17
31.7 keV (x-ray-K v) 35.4 keV (unconverted)
4.5 7,0
26.3 keV (KLM)
7
3.0 keV (LMM) 3,5 keV (LMN) 0,5 keV (MNN)
107 54 265
0.5 keV (MNO)
34
The increased efficacy of 12-sl when incorporated into DNA has been attributed to the severely constricted distribution of the energy of decay resulting from multiple electrons, with a high possibility of damage to both strands of the DNA. ~5't6 Seventy percent of the electrons have initial energies of less than 0.5 keV and produce, on the average, one ionization per 20 ~ in unit density material. Another possible explanation for the greater effect of '25I-IUdR could be disruption of the molecule during the decay process, which produces a heavily positive-charged tellurium atom) v This is a highly unstable situation, in which electrons are attracted from other atoms in the molecule, as demonstrated by Carlson and White L~and Wexler ~9in organic molecules in the gaseous phase. Hofer, Keough, and Smith investigated this possibility and showed, by a double-labeling technique, that molecular fragmentation is probably not a factor in a cellular system. ~5 Column chromatography was carried out on a sample of 14C-t2~IUdR incubated for 76 days at 4 ~ C. The principal effect was noted to be deiodination, with all the t4C contained in a single peak of the eluted material. Furthermore, it was estimated that deiodination occurred with virtually 100% efficiency. The increased biologic effect of ,25| is entirely a consequence of its intranuclear localization when labeled to deoxyuridine. The intense,
124
HAROLD L. ATKINS
I00
[I
~ =
ii
90- 1 8O70-
6050Z o
I--
40-
30N N
aH
m
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1261
|@ - - 0
I I0
[---20
T
50
I
40
J
50
l
60
d/cell/hr
extremely localized ionization events must involve the genetic apparatus in the cell nucleus. Iodine 125 irradiation of the cell membrane and cytoplasm (by labeled concanavallin A, Fig. 4) is no more effective than other forms of radiation in causing loss of reproductive integrity. 2~ This theory is strengthened by studies on V79 Chinese hamster cells in vitro. The efficacy of cell killing corresponded to ability to induce chromosome breaks in a comparison of ~25IUdR, 13tIUdR, and 3HTdR 2~ (Fig. 5). Application of ~2SlUdR to clinical cancer therapy is not a trivial problem. Intravenous administration does not lead to selective localization, and rapid deiodination occurs. Uptake in cells occurs only during the DNA synthesis phase of cell growth. Nevertheless, the high efficiency of I25IUdR in producing cell death has stimulated some investigation into its possible use in cancer therapy. An ascites tumor model in the C3HeB/FeJ mouse has been investigated] 2-25 Intraperitoneal
~
70
t
80
Fig. 3. Fraction of L1210 cells surviving various radioisotope doses, plotted as a function of disintegrations/cell-hr. The lack of a shoulder to the 1251 curve is characteristic of high LET-type radiation (reproduced with permission14).
administration of the 125IUdR permits direct access to the tumor cells. Fractionated therapy was used in order to expose all cells during the DNA synthesis phase. As in other systems studied, an increased efficacy of ~25IUdR as compared to ~3~IUdR was demonstrated (Fig. 6). Application to human cancers is not immediately obvious. Possibilities are suggested, however. Perhaps direct intraarterial administration to tumor sites might be efficacious when dehalogenation beyond the tumor protects the rest of the body. Other agents with high nuclear affinity should be sought. Given enough concentration of a high capture cross-section nuclide, the possibility for neutron capture therapy exists. SELECTIVE IRRADIATION WITH ASTATI N E-21 1
There has been a recent renewal of interest in the chemistry of astatine, with the hope of incorporating astatine 211 into organic molecules in
POTENTIAL FUTURE APPLICATIONS
125
/251UdR
6 4
'-,~
i
2 0
"M
L ~ ___
'_L~_
'
_~___
I
/s/IUdR
% \
i~~~brone 125I
oz~u 0 Hours 9 9 Hours
4 12
~
-
A
~
z~
JllrdR
6
D
E3
E3
9
E3 r
,,\
~
0
9
0.5
f.O
Dc~ ia
J f.5
I 20
j
2.5
pC~/CELL ,
I
800 DOSE (rod)
i
I
1200
Fig. 4. Fraction of Chinese hamster ovary cells surviving various doses of 12SI-UdR (nucleus), 3H-TdR, or concanavallin A 1251(membrane), plotted as a function of radiation dose to the cell nucleus (reproduced with permission2~
order to accomplish selective radiation of specific cellular types or organs. Decay of 7.2-hr 2~At occurs with emission of a 5.87-MeV alpha particle 42% of the time. The other 58% of decays are by electron capture to 0.5-sec 2~Po, which in turn emits 7.45-MeV alpha particles. 26 Astatine belongs to the halogen group, with some similarities to iodine. The biologic distribution of this element was studied almost immediately after its first production in 1940. 27 Uptake in the thyroid was lower than for radioiodine, but selective localization in the thyroid was definitely apparent. The destructive action of the densely ionizing, high-LET alpha particles was demonstrated in histologic examination of rats that had received various amounts of 2~AT.2~ Observations were also extended to patients receiving 2~JAt preoperatively by oral administration. 29 Thyroidal uptake was as great as 17%, and the degree of uptake appeared to correlate with iodine content. Prior administration of
Fig. 5. Total chromosome breaks (chromatid and chromosome type) induced in V79 Chinese hamster cells immediately (0 hr) or 24 hr after removal of lZSIUdR, lzllUdR, or 3H-TdR. The lines are drawn by eye-fit (reproduced with permission 21), 1311UdR
,-~i i~ 10-2 2 ~ 10_3.
~2sIUdR
io-4 i
10-5[
0
Fig. 6.
L gO
L I 1 I 40 60 80 I00 /z,' d'//DOSE, q 4 h x 4
Dose-response curve for 12SlUdR and ?3~IUdR
therapy in a mouse ascites tumor. Animals were treated every 4 hr for four doses, starting 24 hr after the i.p. injection of 10 s tumor cells (reproduced with permission23).
126
HAROLD L. ATKINS
stable iodine decreased uptake. Concentration in papillary adenocarcinoma contained in a lymph node was very low. While accumulation of astatine may be increased in the thyroid by prior administration of agents that block organification of iodine, the application of 2'~At to therapy of thyrotoxicosis or thyroid malignancy has not been accomplished. It seems unlikely that radioastatine will be used for this purpose. Of more recent interest has been the possibility of labeling various organic molecules with 2~At for the purpose of selective destruction of specific cells, while sparing nearby cellular elements that do not accumulate the compound. Hughes and Gitlin 3~ as far back as 1955 attempted the labeling of proteins with 21~At, but the products were not stable. The usual methods utilized in radioiodine labeling have not yielded good results with astatine. Electrooxidation and the use of hydrogen peroxide in the presence of 0.2 #M KI have produced yields of 30%-60% in labeling of proteins. 3~ Zalutsky et al. 32 have accomplished stable binding of astatine to bovine serum albumin, with 12% yield. The labeled material retained its immunospecific properties. Labeling was carried out in a two-step process, first forming an astatinated benzoic acid and then conjugating this to the serum albumin. Another method of labeling with 2~At has been via the decomposition of 5-diazonium salts in the presence of ionic astatine to form 5astatouracil and 5-astatodeoxyuridine. 33'34 Yields of 30% have been obtained. Animal distribution studies in normal and tumor mice have been carried out. 34 The astatinated compounds appear to have a greater concentration in tumor tissue than the corresponding iodinated compound by a factor of 3.
A
B
~lprn
Further work is being carried out to establish the biologic effects and distribution of astatinelabeled compounds. Possibilities include tumorseeking materials, labeling of immunospecific proteins, and destruction of lymphocytes for prevention of transplant rejection. 35'36 LIPOSOMES AS CARRIERS OF THERAPEUTIC AGENTS
Research into the properties of lipid bilayers has resulted in the continuing development, during the past decade, of multilamellar vesicles or liposomes as carriers to deliver biologically active materials into cells. Liposomes are formed by evaporating to dryness a solution of lipid in an organic solvent and then dispersing the resulting film of lipid in water or aqueous buffer. 37'38 Multilamellar concentric bilayer vesicles are formed, consisting of alternating layers of lipid and aqueous medium. Sonication may be used to decrease the size of liposomes. Size may range from 25 nm to 1 #m (Fig. 7). Various materials, such as large proteins, enzymes, radiopharmaceuticals, and chemotherapeutic agents, can be entrapped in liposomes. The degree of entrapment is low, and separation of the liposomes from the free solution is necessary. This may be accomplished by gel filtration, centrifugation, and dialysis. The possibility of using liposomes to deliver therapeutic materials to cells has a number of advantages. 39 First, the biologically active material is protected from degradation in the blood. In addition, the liposomes are able to carry the material across the cell membrane and into the cytoplasm, where the subsequent release of the entrapped material can affect cell metabolism. By the use of various methods of targeting the liposomes, the active material can be directed to specific locations, thus sparing other tissues from
Lipid Biloyer
tarnellae
C
,~_..~
Aqueous Spaces
_ ~ 25 nm
Fig, 7. Schematic representations of liposomes. (a) Multilamellar liposome. (b) Enlarged view of (a). (c) Small unilamellar liposome (reproduced with permission37).
POTENTIAL FUTURE APPLICATIONS
127
the biologic effects. Delayed elimination and decreased chances for allergic reactions are characteristic of liposome-entrapped drugs. The distribution of liposomes administered i.v. is primarily to the reticuloendothelial system. Incorporation into cells may be by endocytosis or by fusion with the cellular plasma membrane4~ (Fig. 8). Distribution may be affected by liposome size, charge, or incorporation of immunospecific globulins. Disappearance from the blood is fairly rapid during the first 2 hr, but a significant proportion of the injected dose may still be circulating several hours after administration (Fig. 9). Slower clearance is evident for the smaller liposomes. Much of the interest in liposomes is related to the delivery of chemotherapeutic agents in the treatment of malignancy and enzymes in deficiency and storage disorders. Research into the application of this system to the use of radionuclide therapy is limited. Radioactivity may be added to liposomes by including the labeled compound in the aqueous medium that is used to prepare the liposomes. Labeling may also be accomplished after formulation of the liposomes. For example, entrapped albumin has been labeled with radioactive iodine using the monochloride method4~ and bleomycin has been labeled with radioactive indium.4] Nucleus
i~
kysosome /
mamembrane
~. Drug-containing liposome Fig. 8. Cellular uptake and lyzosomotropic action of liposome-entrapped drugs. The liposome is taken up by endocytosis. The endocytic vacuole fuses with a lyzosome, the hydrolases of which disrupt the lipid bilayers of the liposome, releasing the entrapped drug. The drug is then able to diffuse out and act in other cellular compartments (reproduced with permission4~
40, 30q
20 x
~8
; ~.
8
7 5 84
2 9
1
2
3
&
4
5
6
Daysafter Injection Fig. 9. Trichloroacetic acid-precipitable radioactivity in plasma of three patients injected with liposome-entrapped lZ'l-labeled albumin (reproduced with permission4~
Labeled antibodies and other proteins may be attached to the outer membrane. 42 Liposomes were labeled with 99mTcusing a SnCI 2 reduction method, by Richardson et al., with 97% efficiency.43-45The SnCI 2 was added to the preformed liposomes, followed by a pertechnetate solution. Distribution studies in rats were performed by imaging of and killing of the animals after administration of positive, negative, and neutral liposomes. Negatively charged liposomes had the slowest blood clearance, followed by positively charged and neutral ]iposomes. Distribution in organs at 29 hr was similar for positive and neutral liposomes, with the major portion of the administered radioactivity in liver (~24%), spleen (~17%), stomach and intestines (~7-8%), and kidneys (~9%). Negatively charged liposomes demonstrated somewhat less liver uptake (11.88%) and more kidney uptake (11.06%). Tumor-bearing (Walker 256 carcinoma) rats were also studied, and demonstrated a similar pattern of distribution. It was possible to image tumors at 12 hr with positive and negative liposomes (Fig. 10). Tumor uptake of negatively charged liposomes could be increased by a factor of 7 with extensive sonication. Maximum tumor
128
HAROLD L. ATKINS
TUMOUR
C
B
TUMOUR
TUHOUR
Fig. 10. Computer-drawn contour mapping of 99mTc in rats bearing Walker 256 carcinoma. (a) S9mTcStannous colloid. (b) Anionic liposomes. (c) Neutral liposomes. (d) Cationic liposomes. The tumor is best outlined with anionic liposomes labeled with 99"Tc (reproduced with permission4S).
concentration was 1.3%/g compared to liver concentration of 1.62%/g, spleen concentration of 3.96%/g, and kidney concentration of 8.72%/8. The same investigators also studied twelve patients with an assortment of malignant tumors. 44 In none was significant localization in tumor apparent by imaging procedures. Liver and spleen uptake were readily noted, but no uptake was observed in two patients with hepatoma. In some patients there was marked uptake in the bone marrow. This was especially apparent in one patient with polycythemia vera, in whom there was very little liver uptake. In another study involving human patients, Gregoriadis et al? ~ were able to obtain tissue samples in two of three patients administered t3q-human serum albumin-containing liposomes. Examination of the tissues at autopsy 5 days after administration of the liposomes was carried out in a patient with transitional cell carcinoma of the bladder, with liver and spleen metastasis. In this patient, 81% of the radioactivity injected was found in the liver. The primary tumor in the kidney had 50 times the concentration of radioactivity of normal kidney, but the differential between normal liver and liver metastasis was negligible. Of interest was evidence of lack of catabolism of the labeled albumin in the primary tumor. This was not true for metastatic deposits or normal tissue. The other patient had a resection of a colon
tumor that had metastasized to the liver. Tissue samples were obtained 3 hr after injection. Again, a negligible difference was found in liver metastasis and normal liver, while the colon tumor had slightly more than two times the concentration of radioactivity of normal colon. Lymph node uptake of neutral or positively charged liposomes has been noted after interstitial administration of liposomes labeled with 99mTC.44
To date, the major interest and possibilities of liposomes are related to the delivery of biologically active compounds to cells. 46 52 The use of radioactive materials has been extremely limited, and there would seem to be little advantage to the use of radiation over chemotherapeutic modalities. Much investigation is needed to improve the specific targeting of liposomes. An example is the attachment of immunospecific molecules on the liposome surface. 42 When this is achieved, it may be possible to incorporate alpha-emitting or Auger electron-emitting radionuclides in order to provide high, LET-type radiation limited to specific cell types. An additional possibility is the e n t r a p m e n t of highcapture, cross-section nuclides, which could be delivered to tumors or organs for exposure to a neutron beam for localized radiation effects. ACKNOWLEDGMENT
The assistance of Drs. Ursula Reincke, Medical Department, Brookhaven National Laboratory, and Clifton R. Harris, Harvard Medical School, is greatly appreciated.
POTENTIAL FUTURE APPLICATIONS
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REFERENCES
1. RP Spencer (ed): Therapy in Nuclear Medicine. New York, Grune & Stratton, 1978 2. Reincke U, Burlington H, Cronkite EP, et al: Selective damage to erythroblasts by ~Fe. Blood 45:801 810, 1975 3. Reincke U, Burlington It, Cronkite EP, et al: Hayflick's hypothesis: an approach to in vivo testing. Fed Proc34:71 75, 1975 4. Reincke U, Brookoff D, Burlington H, et al: Susceptibility of hematopoietic stem cells (CFU-s) to 55Fe radiation damage. Radiat Res 74:66 78, 1978 5. Reincke U, Brookoff D, Cronkite EP, ct al: Relevance of specific activity in experimental crythrocytocidc by 55Fc. Experientia (in press) 6. Laissue JA, Burlington H, Cronkite EP, et al: Induction of osteosarcomas and hemalopoietic neoplasms by ~SFe in mice. Cancer Res 37:3545 3550, 1977 7. Thomson RAE, Corriveau O J, Rubin P: 59Fc labelling inbone. J N u c l M e d 15:161 163, 1974 8. Ertl HH, Feinendegcn LE, tteiniger H J: Iodine-125, a tracer in cell biology: physical properties and biological aspects. Phys Med Biol 15:447 456, 1970 9. Lewitus Z, Lubin E, Rechnic J, et al: Treatment of thyrotoxicosiswith~251and~l. SeminNuclMed 1:411 421, 1971 10. Siemsen J, Wallack MS, Martin RB, et al: Early results of ~251therapy of thyrotoxic Graves" disease. J Nucl Med 15:257 260, 1974 11. Weidingcr P, Johnson PM, Werner SC: Five years" experience with iodine-125 therapy of Graves's disease. Lancet 2:74-77, 1974 12. Glanzmann C, Horst W: Ergcbnissc dcr Therapie der Hyperthyreoscmit 125-Jod. NuclMcd(Stuttg) 14:207 218, 1975 13. Gimlctte TMD, Hosehl R: Treatment of thyrotoxicosis with iodine-125 in moderately low dosage. Clin Radiol 24:263 266, 1973 14. Hofer KG, Hughes WL: Radiotoxicity of intranuelear tritium, ~"Siodineand ~3~iodine. Radiat Res 47:94 109, 1971 15. Hofer KG, Keough G, Smith JM: Biological toxicity of Auger emitters: molecular fragmentation versus electron irradiation. CurrTopRadiat Res. 12:335 354, 1977 16. Feinendegen LE: Biological damage from the Auger effect, possible benefits. Radiat Environ Biophys 12:85 99, 1975 17. Carlson TA, White R M: "Explosion" of multicharged molecular ions: chemical consequences of inner shell wlcancies in atoms, in: Chemical Effects Associated With Nuclear Reactions and Radioactive Transformations. Proceedings of a Conference, vol I. Vienna, IAEA. 1965, pp 23 33 18. Carlson TA, White RM: Formation of fragment ions from Ctt~Te ~25 and C2H~Tc125 following the nuclear decays ofCH~l ~'~ and C2H~112~. J Chem Phys 38:2930 2934, 1963 19. Wexler S: Destruction of molecules by nuclear transformations. Science 156:901 907, 1967 20. Warters RL, Hofcr KG, Harris CR, et al: Radionuelide toxicity in cultured mammalian ceils: elucidation of the primary site of radiation damage. Curr Top Radiat Res 12:389 407, 1977 21. Chan PC, Lisco E, lfisco H, ctal: The radiotoxicity of iodine-125 in mammalian cells. 11. A comparative study on
cell survival and cytogenetic responses to ~251UdR, ~3qUdR, and 3HTdR. Radiat Res 67:332-343, 1976 22. Bloomer WD, Adelstein S J: Therapeutic application of iodine-125 labelled iododeoxyuridine in an early ascites tumor modeI. CurrTopRadiatRes12:513 525, 1977 23. Bloomer WD, Adelstein S J: 5-(t2~l)-iododeoxyuridine in experimental tumor therapy, in Spencer RP (ed): Therapy in Nuclear Medicine. New York, Grune & Stratton, 1978, pp 177-183 24. Bloomer WD, Adelstein S J: Antineoplastic effect of iodine-125-1abelled iododeoxyuridine. Int J Radiat Biol 27:509-511, 1975 25. Bloomer WD, Adelstein S J: 5J251-iododeoxyuridine as a prototype for radionuclide therapy with Auger emitters. Nature 265:620-621, 1977 26. Lederer CM, Hollander JM, Perlman 1: Table of Isotopes (ed 6). New York, Wiley, 1967 27. Hamilton JG, Soley MH: A comparison of the metabolism of iodine and of element 85 (eka-iodine). Proc Natl AcadSciUSA26:483 489, 1940 28. Hamilton JG, Asling CW, Garrison WM, et al: Destructive action of astatine TM (element 85) on the thyroid gland ofthe rat. ProcSocExpBiolMed73:51 53, 1950 29. Hamilton JG, Durbin PW, Parrott MW: Accumulation of astatine 2~ by thyroid gland in man. Proc Soc Exp Biol Med 86:366 369, 1954 30. Hughes WL, Gitlin D: Astatinated (At 2~) organic compounds for production of highly localized radiobiological effects. Fed Proc 14:229, 1955 31. Aaij C, Tschroots WRJM, Lindner L, et al: The preparation of astatine labelled proteins, lnt J Appl Radiat Isot26:25 30, 1975 32. Zalutsky MR, Friedman AM, Buckingham FC, et al: Synthesis of a non-labile astatine-protein complex. J LabelledCompRadiopharm 13:181 182,1977 33. Meyer G J, R6ssler K, St6cklin G: Preparation and high pressure liquid chromatography of 5-astatouracil. J Labelled Comp Radiopharm 12:449-458, I976 34. R6ssler K, Meyer G3, St6cklin G: Labelling and animal distribution studies of 5-astatouracil and 5-astatodeoxyuridine (2~At)..I Labelled Comp Radiopharm 13:27 I, 1977 35. Myburgh JA, Smit JA: Enhancement and antigen suicide in the outbred primate. Transplant Proc 5:597-600, 1973 36. Smit JA, Myburgla JA, Neirinckx RD: Specific inactivation of sensitized lymphocytes in vitro using antigens labelled with astatine-211. Clin Exp lmmunol 14:107 116, 1973 37. Tyrrell DA, Heath TD, Colley CM, et al: New aspects of liposomes. Biochim Biophys Acta 457:259 302, 1976 38. Poste G, Papahadjopoulos D, Vail W J: Lipid vesicles as carriers for introducing biologically active materials into cells. Methods Cell Biol 14:33 71, 1976 39. Gregoriadis G: Targeting of drugs. Nature 265:407 41 I, 1977 40. Gregoriadis G, Swain CP, Wills EJ, et al: Drugcarrier potential of liposomes in cancer chemotherapy. Lancet 1:1313 1316, 1974
130
41. Neerunjun ED, Hunt R, Gregoriadis G: Fate of a liposome-associated agent injected into normal and tumourbearing rodents: attempts to improve localization in tumour tissues. Biochem Soc Trans 5:1380-1383, 1977 42. Gregoriadis G, Neerunjun ED: Homing of liposomes to target cells. Biochem Biophys Res Commun 65:537-544, 1975 43. Richardson V J, Jeyasingh K, Jewkes RF, et al: Properties of (99mTC)technetium-labelled liposomes in normal and tumour-bearing rats. Biochem Soc Trans 5:290-291, 1977 44. Ryman BE, Jewkes RF, Jeyasingh K, et al: Potential applications of liposomes to therapy. Ann NY Acad Sci 308:281-307, 1978 45. Richardson VJ, Jeyasingh K, Jewkes RF, et al: Possible tumor localization of Tc-99m-labelled liposomes: effects of lipid composition, charge, and liposome size. J Nucl Med 19:1049-1054, 1978 46. Chen JS, Del Fa A, DiLuzio A, et al: Liposome-
HAROLD L. ATKINS
induced morphological differentiation of murine neuroblastoma. Nature 263:604-606, 1976 47. Juliano RL: The role of drug delivery systems in cancer chemotherapy. Prog Clin Biol Res 9:21-32, 1976 48. Neerunjun ED, Gregoriadis G: Tumour regression with liposome-entrapped asparaginase: some immunological advantages. Biochim Soc Trans 4:133-134, 1976 49. Colley CM, Ryman BE: The use of a liposomally entrapped enzyme in the treatment of an artifical storage condition. Biochim Biophys Acta 451:417-425, 1976 50. Kimelberg HK: Differential distribution of liposomeentrapped (3H) methotrexate and labelled lipids after intravenous injection in a primate. Biochim Biophys Acta 448:531-550, 1976 51. Fendler JH, Romero A: Liposomes as drug carriers. Life Sci 20:1109-1120, 1977 52. Gregoriadis G: The carrier potential of liposomes in biology and medicine. N Engl J Med 295:765-770, 1976
