as hereinbefore stated , the use of alpha emitters provides substantial advantages in the treatment of cancer . it is also possible to treat inflamed joints particularly rheumatoid arthritis with radioisotopes . for the reasons that alpha emitters are useful in the treatment of cancer , they are also useful in the treatment of arthritic joints . radiation synovectomy has substantial advantages over the treatment by surgery , not the least which is that it can be repeated if the condition reoccurs . other therapies often result in the eventual replacement of the joint . as before stated , radiation synovectomy suffers from leakage of the radioactive material from the joint . the use of alpha emitters which have a very short range of effectiveness is somewhat helpful in alleviating this condition , particularly when combined with the carrier hereinafter described . colloids labeled with radionuclide have been used in the past for the treatment of cancer and also for the treatment of rheumatoid arthritis . because the alpha emitters are effective over a relatively short cellular range , labelling thereof with colloids has not been popular . this is particularly true where the radionuclide normally would be distributed evenly throughout the colloid because the distance that the alpha particles travel from the center of the colloid to the outside of the colloid is such that very little effective radiation remains to be delivered to the affected situs . the use of colloids labeled with alpha emitting radionuclides are advantageously employed both for treating cancer cells and also for inflamed joints provided that there is some mechanism for plating the radionuclide essentially on the outer surface of the colloid and only on the outer surface . it has now been discovered that ferric hydroxide can be manipulated in such a way as to plate the radionuclide essentially on the outer surfaces of the colloid . radionuclides which are useful in the present invention include 211bi , 212bi , 213bi , 214bi , 212pb , 228th , 224ra , 211at , 254esm , 238np , 234np , 242am and various mixtures thereof . many of these materials are produced in reactors or cyclotrons , but it is well within the skill of the art to manufacture and isolate the above listed alpha emitters . for instance , the preparation of various bismuth isotopes as well as the lead - 212 is disclosed in the atcher et al . u . s . pat . no . 4 , 663 , 129 , issued may 5 , 1987 , the disclosure of which is incorporated herein by reference . methods of producing and separating astatine - 211 as well as certain bismuth radioisotopes has been reported in the literature , see for instance appl . radiat . isot . vol . 39 . no . 4 , pp . 283 - 286 , 1988 , reporting a paper on radiation oncology . see also , official journal of the american rheumatism association , arthritis and rheumatism , vol . 29 , no . 2 , february , 1986 in which the use of beta emitters for treatment of arthritic joints has been reported . the colloid of the present invention must be prepared in such a manner that rather than having the radioactive isotope uniformly distributed throughout the colloid as is the usual circumstance , the radionuclide is essentially only on the outer surfaces thereof . in order to make this particular colloid , a ferrous salt such as a sulfate is treated with a hydroxide such as ammonium hydroxide to provide ferrous hydroxide . after the addition of radioactive 212pb in the form of lead iodide , the ferrous hydroxide lead iodide mixture is mixed in air to oxidize the ferrous hydroxide to a ferric hydroxide colloid in which the lead isotope is attracted to and plates onto the outside surface of the colloid particles . the ferrous hydroxide may be formed either by starting with ferrous chloride or ferrous sulfate in an acid solution , such as a dilute hydrochloric acid . a suitable hydroxide such as ammonium hydroxide in reagent grade is used to convert the ferrous salt to the ferrous hydroxide . a vortex mixer is used to convert the ferrous hydroxide which does not attract the lead iodide in solution to ferric hydroxide which does attract the lead iodide in solution , the ferrous ion also converting the lead iodide to lead metal . in a specific example , ferrous chloride at a concentration of 5mg / ml in dilute hydrochloric acid is mixed with a 2 molar solution of hydriodic acid containing lead212 . reagent grade ammonium hydroxide , 14 . 5 molar , is added to the mixture to form a bluish green ferrous hydroxide precipitate in a solution with the ph greater than 7 . at this time the lead iodide remains in solution and is not attracted to the ferrous hydroxide particles . an air vortex mixer is used to agitate the ferrous hydroxide and lead iodide solution for approximately 30 seconds whereupon the bluish - green mixture disappears to be replaced by a dark yellow - brown mixture of ferric hydroxide which attracts the 212pb to the surface principally as a metallic lead although a portion of the lead is present as lead hydroxide . this method of forming a colloid results in a ferric hydroxide colloid having radioactive 212pb on the outer surface of the colloid either as lead metal or lead hydroxide and since the lead daughter , 212bi , is an alpha emitter , provides a colloid with the most effective positioning of the radioisotope . to study in - vivo the therapeutic effect of the intraperitoneal instillation of the radiocolloid , 212pb ferrous hydroxide , the ehrlich ascites carcinoma model was used . this carcinoma spontaneously arose in the ovary of the mouse . the carcinoma has been maintained by intraperitoneal inoculation and passage in swiss - webster mice . the virulence of the tumor was evaluated by inoculating groups of 10 mice ip with 10 0 to 10 7 cells . survival was then measured from the day of inoculation . the therapeutic effect of ip administration of 212pb ferrous hydroxide was evaluated by treating groups of 10 animals inoculated with 106 cells with single graded doses of 0 , 5 , 15 , and 50 uci &# 39 ; s and measuring survival . the effect of delaying therapy was determined by observing survival in groups of 10 animals with 10 6 cells treated with 50 uci &# 39 ; s of the radiocolloid 48 and 72 hours later . the cytotoxicity of 212pb was compared to x - rays . ehrlich carcinoma cells were grown in - vitro in 5cc of serum culture containing 72 . 5 % dulbecco &# 39 ; s modification of eagle &# 39 ; s media , 22 . 5 % ham &# 39 ; s nutrient mixture f - 12 , 5 % fetal bovine serum , 20 mg / ml epidermal growth factor , 5 ug / ml transferrin , 2 × 10 - 11 m 3 , 3 &# 39 ;. 5 triiodo - 1 - thyronine , 10 - 10 m cholera toxin , 1 . 8 × 10 - 4 m adenine , 0 . 4 ug / ml hydrocortisone , 50 units / ml mycostatin , 100 u / ml penicillin , and 100 ug / ml streptomycin . survival experiments were done on exponentially growing cells . cells were removed from flasks using trypsin suspended in serum containing media and seeded into 10 cm dishes at low density . between 500 and 40 , 000 cells were plated and allowed to enter exponential growth . to determine cellular survival after 212pb irradiation , the radionuclide complexed to dpta was diluted in complete culture medium . the activity of an aliquot was determined by counting the gamma rays in a spectrometer which was calibrated with a 228th source . cells were incubated at 37 ° c in media containing various radioactive concentrations of 212pb dpta . after the appropriate dose accumulated , the cells were washed and fed fresh media . control incubations were done in an identical fashion except the 212pb was replaced by 212pb which had decayed to determine chemical toxicity . for x - ray survival experiments cells were irradiated 18 hours after plating with a 250 kv maxitron operating at 26 ma ° at 0 . 8 gy / min . cultures were incubated for 18 to 24 days and then fixed and stained with crystal violet . colonies greater than 50 were scored as survivors . data points were analyzed by least square regression . the intrinsic radiosensitivity ( do ) was defined as the inverse of the slope of the exponential portion of the survival curve . the cell s ability to accumulate sublethal damage was measured by the extrapolation number , n , which is the back extrapolation of the slope of the ordinate . the ehrlich carcinoma cells were extremely virulent . the intraperitoneal injection of graded doses from 10 0 ( 1 cell ) to 10 7 cells caused ascites leading to the death of the animal within 57 to 58 days , respectively ( table 1 ) a tumor inoculum as small as 1 cell caused death in 80 % of the animals . treating animals 24 hours later inoculated with 10 6 tumor cells with graded doses of 212pb ferrous hydroxide prolonged survival . in the untreated inoculated animals the mean survival was 16 days . the mean survival after the injection of cold colloid along , 5 , 15 , or 50 uci &# 39 ; s of the radiocolloid was 15 , 49 , 63 , and 81 days , respectively ( table 2 ). the percentage of animals cured was related to the dose of the radionuclide administered . the cure rate was 0 , 10 , 23 and 40 %, respectively for the doses administered . delaying therapy to allow the tumor to progress decreasing survival . the mean survival in animals inoculated with 10 6 cells decreased to 45 and 34 days by delaying treatment 48 or 72 hours , respectively . in - vitro the ehrlich cells were more radiosensitive to alpha particles than x - rays ( table 3 ). the survival curve had a steeper slope after 212pb therapy . the radiosensitivity ( do ) was 220 cgy after x - ray and 65 cgy after 212pb irradiation . cells which were able to accumulate sublethal damage after x - rays were unable to do so after 212pb irradiation . there was a shoulder ( n = 1 . 7 )- an indication of the ability of the cells to accumulate sublethal damage - present on the x - ray survival curve . with 212pb treatment there was no shoulder ( n = 1 ) on the survival curve . the intraperitoneal administration of 212pb prolonged the median survival and produced cures in the ehrlich ascites tumor model . this tumor was extremely virulent with the injection of one cell capable of producing tumor and death in the animals . the survival was dose related with higher doses of 15 and 50 uci &# 39 ; s increasing survival threefold . the total eradication of tumor was seen in 24 % of the animals injected with these doses . the most compelling reason for the increased effectiveness of these particles if the direct ionization over a very short path length without the dependence upon cellular oxygenation for cytotoxicity . the use of these emitters may be most effective against microscopic disease . tumor burden present appears to be an important factor when considering the use of these emitters . by delaying the intraperitoneal instillation of 212pb up to 72 hours and allowing the tumor burden to increase both the survival and cure rates decreased . clinically alpha emitting radionuclides have the potential to be more efficacious than other beta - emitting radionuclides previously used such as gold - 198 and phosphorus - 32 . the cellular radiosensitivity was markedly increased in comparison to conventional gamma ( x - ray ) irradiation . survival was better with cells having no ability to accumulate sublethal danger after x - ray therapy . in comparison to beta - emitters , it is estimated that alpha irradiation has one - hundredth the range and may have up to ten times the energy deposition per unit path length making it more efficient in killing a tumor cell while perhaps sparing normal cells . as seen in the decay chain of 212pb both beta and alpha particles are produced ; however , considering that the total average energy per disintegration of 212pb , the beta energy contribution to the dose is negligible . table 1 . ______________________________________survival of animals inoculated with graded doses ofehrlich ascites tumor cells . number of days survivingcells injected minimum maximum mean % dead______________________________________10 . sup . 7 8 18 14 10010 . sup . 6 12 20 16 10010 . sup . 5 17 29 18 10010 . sup . 4 17 34 22 10010 . sup . 3 19 28 23 10010 . sup . 2 19 30 26 8010 . sup . 1 28 38 33 8010 . sup . 0 28 57 41 80______________________________________ table 2 . ______________________________________survival and cure of animals inoculated withehrlich ascites tumor cells treated 24 hours laterwith lead - 212 ferrous hydroxide . tumor survival ( days ) inoculum treatment min . max . mean % cure______________________________________10 . sup . 6 none 12 20 16 010 . sup . 6 cold colloid 12 22 15 010 . sup . 6 5 uci 212pb 21 150 49 610 . sup . 6 15 uci 212pb 26 150 63 1310 . sup . 6 50 uci 212pb 22 150 81 24______________________________________ table 3 . ______________________________________summary of the radiosensitivities of ehrlichcarcinoma tumor cells to x - rays and lead - 212 . do n______________________________________x - rays 220 1 . 7212pb 65 1______________________________________ do = radiosensitivity n = ability to accumulate sublethal damage rbe = relative biological effectiveness the use of these nuclides have the potential to add another treatment modality for microscopic carcinoma confined to the abdominal cavity . however , the concept is applicable to treatment of other types of carcinoma located in otherwise difficult areas . for instance , tumors of the liver are difficult to treat , but the colloid can be delivered through arterial blood flow to the liver , or for that matter , to any organ . delivery of the labelled colloid to inflamed joints , such as knees , fingers , toes and wrists promises an alternative therapy . while there has been disclosed what is considered to be the preferred embodiment of the present invention , it is understood that various changes in the details may be made without departing from the spirit , or sacrificing any of the advantages of the present invention .
a ferric hydroxide colloid having an alpha - emitting radionuclide essentially on the outer surfaces and a method of forming same . the method includes oxidizing a ferrous hydroxide to ferric hydroxide in the presence of a preselected radionuclide to form a colloid having the radionuclide on the outer surface thereof , and thereafter washing the colloid , and suspending the washed colloid in a suitable solution . the labelled colloid is useful in cancer therapy and for the treatment of inflamed joints .