PH26149A - Perfluorocarbon emulsions and their preparation - Google Patents

Description

. ' 26149

SENSITIZATION OF HYPOXIC TUMOR CELLS

AND CONTROL OF GROWTH THEREDF

RELATED AFFLICAT IONS

This application is a continuation of application

S Ser. No. 724,445, filed Apr. 17, 1985, now abandoned.

This application ie a division of WL.8. application

Ser. No. 5B0O,760, filed Feb. 17, 1284; the latter being : a continuation-in-part of UU. 8. application Ser.

Na. $517,150, filed July 253, 19683, and now abandoned; which in turn was a continuation-in-part of U.S. application Ser. No. 406,589, filed Aug. 17, 1982, and all are now abandoned. Co

FACKEGROUND OF THE ITRVENTION

This invention relates to the sensitization of hyposic tumor cells to therapy, and in particular to methods, compositions and systems for sensitizing hypoxic tumor cells to radiation and/or to certain chemotherapeutic agents, whether the therapy is employed alone or in combination with agents which

L220 protect normal tissues from injury. The invention further relates to diagnostic methods in support of the ‘ sensitization and therapy.

Fo convenience of expression in this specification the following or similar terms are sometimes abbreviated as indicated: "PFC" —~ perfluoro compound "RG" ~ radiosensitization or radiosensitizing "RT" ~ radiotherapy or radiaothesrapeutic 1

BAD ORIGINAL 9

ET" - chemotherapy or chemotherapeutic "RET — radioprotection or radioprotechtive

COTE" - chemotherapeutic protective "RIT —- radioimaging

Ouygen deficient (hypensd a) cells can be up to about three times more resistant te radiation than are wel l-oxigenated cells. These cells are relatively common in tumors bul are rare in normal tissues, thereby giving the tumor cells a greater resistance Lo radiation than one observes in normal tissues. As a consequence, one often cannot deliver enough radiation to eradicate the tumor cells without incurring an unacceptably high risk of severe injury to normal : tissue. These same hypoxic cells are often resistant : 15 to those forms of chemotherapy which are OH YyQgen dependent. in such chemotherapy, however, oxygen must often be supplied to.the hypoxic nells for another - reason in addition to maximal cell destruction: hypoxic cells are not actively growing (multiplying) and many of the more effective chemotherapeutic drugs cannot ill the rells unless they are Qrowing.

Further, hypoxic cells have low energy reserves and are thus helieved less able to actively transport certain chemotherapeutic drugs across their membranes.

The following contribute to the relatively common occurrence of hypoxic cells in tumors: the tumors out- grow their blood supply: blood flow through the vessels in the tumor is sluggishs and the tumor cells near the hlood vessels consume large amounts of oxygen, thereby

. ’ 26149 even further redocing the amount available toy more - distant oells. I4 means could be developed to re- oxygenate these | hyporic areas ons would expect Large increases in radiosensitivity because the hypoxic tumor regions are near the minimam of oxygen dependent radiosensitivity. The same does not hold true for normal tissues, since, under natural conditions, they are already nedr the maximum for oxygen dependent radiosensitivity. The same or similar considerations also apply in sensitizing cells to chemotherapy.

An obvious approach to reversing the resistance of hypoxic cells to treatment is to directly supply the cells with more oxygen. This was initially attempted by injecting hydrogen peroxide which would hopefully release oxygen at the tumor site. The technique has ’ never achieved practicality, however, due to the toxicity of injected hydrogen peroxide.

A potentially less toxic, but similar direct approach, involved having the patient breathe 100% odygen at 3 atmospheres pressure both before and during . radiotherapy. While at least some results were . encouraging, the toxicity of hyperbaric OH ygen treatments has limited the use of this technique to : sub-optimal treatments, ieee, fewer but larger “5 radiatien doses.

A third direct approach was initially tested by

Belgrad et al (Radiology 133: 2305-237, 197G) These investigators oxygen-saturated a pure sample of perfluorooctyl be-omide, Enown as a highly efficient oxygen carrier, and injected the oxygenated, neat = compound into mice bearing the F388 leukemia. Twenty four hours later they sxposed the mice to graded doses af whole body X-rays, and compared the swvival time of these mice to similarly treated mice which had received an injection of a salt solution, The results of these atudies failed to show a significant improvement in } therapy, and local tovic reactions in the peritoneal cavity were observed.

The Relgrad et al study is not useful as a quide ar suggestion of the use of perfluorooctyl bromide or other FFC in hypoxic tumor cell therapy. The study has little clinical relevance, and discourages further studies leading to clinical investigations. One would never consider flooding (as npposed to local administration) the peritoneal cavity with a pure FFG or even a FC in emulsified form. Further, oxygenation ’ of a FFC prior to administration coupled with radiation treatment 24 hows after administration offers almost no opportunity for the leukemic cells to be sensi tized ’ 20 to radiation. Lastly, the highest irradiation doses reported by Relgrad et al are known to be lethal to mice, and thus leave open to conjecture whether the perfluoronctyl bromide had any sensitizing effect at all under such conditions.

In attempts to sidestep oxygen delivery as the primary mode of radiosensitization, radiations have heen used which are less dependent on the oxygenation status of the cell for their cell killing efficiency.

Such radiations are densely ionizing or high LET

. 26149,

Fadiations and have been limited, however, YY tei - unfavorable focusing characteristics. Normal tissues recel ve more of these radiations per unit doses delivered to the tumor than is the case with conventional radiations, ancl this factor has counterbal anced the expected achvantage cute to independence of oxygenation state. Another radiosensitization: teachni que for avoiding gross oxygenation is the usa of drugs which mimic the presence of oxygen but which can diffuse further into the tumor because they are less readily consumed by the cells traversed. Typical of sch drugs are metronidazole, misonidarotle and the nitroimidarole compounds disclosed in U.S. Fat. Nos. 4,241,060 ancl 4,282,232. Such drugs, while initially promising, have not produced large therapeutic gains clinically because the drug doses required to produce significant radiosensitization also produce unacceptable neurotoxicity in patients.

Hypoxia is invariably found in carcinoma and sarcoma but even in benign conditions (where the } hypoxic cell tumors are not continually increasing in mass) , radiotherapy and/or chemotherapy are sometimes prescribed in order to forestall cancerous conditions.

The present invention is therefore applicable both to malignant and genign hypoxic tumor cells.

As indicated in Belgrad et al, perfluorinated hydro-carbons are known which are yr oxygen Carriers and some have been used as blood substitutes.

Nevertheless, this property alone cannot make these oo. 1 neeful as sensitizing agenhs in’ radiotherapy and/ ar oxygen-dependent ~ chemotherapy. In addition to good oxygen transfer capability auch compounds, for effective RS and/or 0TH effect, must: 1. Be capable of rapid transfer to the dense cell populations characterizing hypowic tumor cells or to the vasculature thereof, and of releasing oxygen to these cells; 2. Exhibit favorable residence time in & mammalian system, as opposed to too rapid elimination by excretion, transpiration or metabolism, and as opposed to undue accumulation in the system (as in the liver and spleen); and x. Exhibit no toxicity or tolerable toxicity to normal (euoxic) cells.

Ideally, the oygen transfer compound, if used systematically, will diffuse quickly through the vasculature, pick up oxygen in the lungs, remain the cardiovascular system for about 10 to 12 days, (bo

R20 permit periodic irradiation at controlled dosages) and then be rapidly eliminated, while producing Ne intolerable Loticity. Under such conditions sensitization to radiotherapy and/or chemotherapy Can be maximized. However, residence times AS short as 2 to 8 hours may be sufficient if only short term therapy is necessary. Hence, considerable leeway should be possible in treatment protocols, depending on the types, state of division and site of the tumor cells, and other considerations known to therapists, auch as &

' "E149 type of irradiation or chemotherapeutic agent, side effects, and mode of administration,

SUMMARY OF THE INVENTION

In accordance with the present invenlion effective 3 sensitization of hyporic tumor cells, as a prelude to highly beneficial radiotherapy and/or oxygen-dependent chemotherapy, is achieved by contacting the cells or vasculature therepf with an oxygen carrying perfluoro compound, wherein the perflouro compound is uniformly dispersed in small particle size in an aqueous medium.

The resulting aqueous dispersion is rendered isotonic (or otherwise physiologically acceptable) to mammalian cells prior to use as a sensitizing agent by the addition of salts, buffering agents or other reagents . 15 Ernown to be effective for this purpose.

In one aspect of the invention the dispersion is injected intravenously, is carried through the lungs where it picks up oxygen, and then penetrates the region of the hypoxic tumor cells. The oxygen transferred from the perfluoro compound to the hypoxic cells sensitizes Lhe cells. Simul taneously with bes sensitization or thereafter, the cells are irradiated and/or a OT agent is administered. Cell destruction or reduced rate of growth can be monitored by biopsy, 21 radioimaging or other technique. ("Growth" as used herein means cell multiplications "wankrol of growth or similar term means cell destruction or decreased growbh rate.)

’

D221 AQ9 1 »

In another aspect of the invention, hypoxic tumor rells are oxygenated hyperbarically but at oxygen pressures and/or for periods substantially less savers than conventionally amp loyed for treatment of hypoxia, and contact of the cells lor vasculature Lhereof Wi th the perfluoro compound thus supplements oxygen transfer to the o=lls. This prhanced sensitization is then followed or accompanied by irradiation or chemotherapy in the conventional manner. ‘

In till another (diagnostic) aspect of the invention, penetrabion of the perf luoro compound into the regions of the hypodo tumor ralles is monitored by gsysbematic administration of & preavf Luo o compound which also has radiopacgue properties, thereby permitting radioimaging. a variation of this approach is to incorporate into a dispersion containing an oxygen carrying perf luoro compound which does nok have radioimaging properties, an onother compound which is an RI agent, thereby rendering the hypoxic cells susceptible to radioimaging.

In another aspects of the invention, RF and/or CTF agents are cambined with the oxygen carrying FFC in the dispersion, or are separately delivered to the site of the hypoxia, in order to afford additinnal therapeutic response. This effect is achieved through the ability of the psrfluoro compound to sensitize the tumor cells, while the RF and CTF agents, since they are not absorbed hy the tumor cells, shield the normal tissues } from attack by padiation and the oT agents,

~ , 4 26149 respectively. In some cases the same agent can be berth radioprotective and chemotherapeutic protective.

The term “mammal or similar term as Lasse d throughout this specification is intended in its broad

GS and equivalent sense bo mean and include all animals.

DETATLED DESCRIFTTON

The Sensiltizer Dispersions

The sensitizing agent of the invention is an aqueous dispersion of an oxygen carrying perfluoro compound and a dispersant (surfactant or emulsifier) which is effective for uniformly dispersing the per+luoro compound in the agueous mecliume. The dispersant is required because the perfluoro compounds are relatively hydrophobic and would otherwise tend to agglomerate in the mammalian body fluids through which the compound must pass and which serve to carry the compound to the hypomdc tumor cells or vascular thereof. Thus, although the perfluoro compound in the neat state might initially be injectable into the body, in a short time its tendency to agglomerate would impede its use as a sensitizer. The aqueous dispersion medium also permits addition of reagents for rendering the didpersion isotonic or otherwise physiologically acceptable to the cells. } 25 Generally, the perfluoro compounds and dispersions thereof useful in this invention are those materials identified in the patent and other technical literature as synthetic blood substitutes. Representative of the @

' 26149 patent literature disclosing such hlond substitutes are Co

U.S. Fat. Nos. 3,641,147, 3,823,091, a,11, 138, 3,962,439, X,593,581, 1,041,086, 4,105,798, and 4,325,972, the disclosures of which are incorporated herein by reference.

It will be apparent from a Feaview of the foregoing patents and other literature that a wide variety of perfluorinated compounds when suitably dispersed in an aqueous medium can he uwsed for tha purposes of the present invention. The perfluoro compounds thus include aliphatic (acyclic or cyclic) antd aromatic . compounds, whether perf luocinated ny ocarhons only ov also containing heteroatoms such as oxygen, sulfur and/or ni trogen, and may be used singly or as mixtures af two or more. The selection of perfluoro camp ound for use in specific cases in accordance with the invention will depend on a variety of factors, including whether the treatment is in conjunction with radiotherapy, chemotherapy, ar booths the character ancl locus of the hypoxiag the potency of the per{luoro compound as a sensitizer; toxicity of the perfluaro compound to normal cells and to the host mammals capability of forming sufficiently amall particle size dispersions and sufficiently stable dispersions to diffuse rapidly to the region of the hypouic cells; residence time in the mammal , including accumul ation tendencies; and similar considerations familiar to those knowledgeable in the sensitisation art. Guidance feu much selection oan he obtained from the bh 1 edd 1

' 26149 substitute art, particularly as to oxygen transport capability, dispersion particle size and stability, mammalian residence time, and cytotoxicity.

Additionally, on the basis of the in vitro and in vivo studies reported and discussed hereinafter, guidance is provided for selection of perfluoro compound and treatment parameters in specific cases af sensitization. "fParfluoro compound" or "perfluorocarbon" as used herein refers to a substantially fluorinated or completely Fluorinated material which is generally but not necessarily a liquid at ambient temperature and pressure. "Substantially fluorinated" as used herein means that most of the hydrogen atoms of a compound have been replaced by fluorine atoms, such that further replacement does not substantially increase the onygen transport capability of the material. It is believed that this level is reached when at least B0-90% of the hydrogen atoms have been replaced by fluorine atoms.

However, it is preferred that at least 95% of the hydrogen atoms have been replaced, more preferably at least 98% and most preferably, 100%. In the afore- mentioned U.S. Fat. Nos. 3,911,138 and 4,105,798, the ability to transport oxygen is related to the solubility in the materials of a gas such as oxygen.

These patents suggest that the perfluorinated materials will absorb 10-100 cc of oxygen per 100 cc of material at —. and T60 milliliters of mercury. ‘ Representative of the perf luoro compounds preferred for use in this invention are the

: C ] 261490 perfluorinated derivatives of chemically inert © -C gf 18 polycyclic compounds such as bicyclononanes (e.d., bicyclol3.3.1Inonane, 2,6-dimethylbicyclol3. 3.1 Inonane,

A—methylbicyclol3. 3. 1dnonane and trimethylbicyclof3.3.173- nonane) adamantane and alkyl (CC —-C ) adamantanes such as methyl and di mathyl adamantane, ethyl and diethylada-— mantane, trimethyladamantane, ethylmethyl adamantane, ethyldimethyladamantane and triethyladamantanes methyldiadamantane and trimethyldiadamantane; methyl and dimethylbicyclooctanes; tetrahydraobinor-§8, pinane, camphane, decalin and alkyl decalins such as 1-methyl~ decalin: and 1,4,6,9-dimethanodecaling bicyclol4.3.21- uwndecane, bicyclolS. 3. 0ldecane, bicyclol2.2.1loctane, tricyelol5. 2.1.0 | Jdecans, nothyltricyelofs. 2.1.0 | 1- decane, and the like; or any mistures thereof. Hetero atom perfluoro compounds include F-tributyl amine,

F-~tripropyl amine and F-~N,N~dimethylcyclohexylmethyl- amines perfluoro ethers such as F-2 -butyltetrahydro- furan, F-2-butyl furan, F-hydrofuran, the 1,2,2,2,~ tetrafluoramethyl ether of F-(2,5,8-trimethyl-3,6,%- trioxa-i-dodecanol), F-M-methyldecahydrogquinal ine, F-1- methyloctahydroquinolizine, F-octahydroguinolidine and

F-N-cyclohexylpyrrolidine. Aromatic and aliphatic compounds include F-naphthalene, F-1-methylnapthalene,

F-n-methyl-morphaline, F-n—-heptane and 1,2-bis-nonyl- fluorohutylethylene.

Certain of the fluorine atoms of the foregoing materials may be substituted by other halogen atoms such as bromine. Included among these compounds, are,

for example, monobrominated Lompounds such as 1- bromopentadecafluoro-4-isoprapylcyclohexane, 1--bromotrideca- fluoro-hexane, l-bromo-pentadecafluorooctane and 1-bromo- pentadecafluoro-3-isopropyl-cyclopentane and perfluoro- 1-bromobutylisopropyl ether, or polybrominated derivatives thereof.

When bromo or iodo atoms appear in the perfluoro compounds, the compounds tend to be radiopaque while also retaining a large measure of their oy gen transporting capabilities. The radiopacity renders these compounds useful as radioimaging (RID agents, and therefore these compounds in some cases can be used not only as sensitizing agents but also as RI agents, alone or in combination with other sensitizers and/or RI agents.

It is known that the rate of transpiration of perfluorinated hydrocarbons from lower mammals is in . the order: tricyclics»*bicyclicsralkyl monocyclics»>— paraffinics. Accordingly, where high rate of transpiration is preferred, for example when only a brief interval of irradiation is prescribed, a ’ tricyclic perfluaro compound will be preferred over other perfluoro compounds. Conversely, if extended radiotherapy or chemotherapy is desired, therefore requiring longer sensitizer residence time, a bicyclic or monocyclic perfluoro compound might be chosen.

The more preferred perfluoro compounds far use in the invention on the basis of relative inertness (chemical and biological), good dispersability and

" 26149 residence time are the perfluoro C -C polycyclic 7? 18 hydrocarbons of u. 5. Fat. No. 4,105,798, and particularly F~dimethyl adamantane, F-trimethylbicyclo- nonane, FetricyclolS. 2. 1.0 ldecana, F-methyltricyclo- 2 (5.2.1.0 Jdecane, F-bicyclolS.2.0]decane and

F-methylbicyclold.2.0)decane, including any isomers thereof, and mixtures of such compounds, for example mixtures of F-dimethyl adamantane and Fo trimethylbicyclononane, ranging from about F0/10 to 10/920 by weight.

The preferred dispersants for unifarmly dispersing the perfluoro compounds in an agueous medium are the nonionic surfactants. In some compositions and systems of the invention, particularly those cases where the dispersions are used non—-systematically, such as in topical or local treatments, ionic or amphoteric surfactants may be used to disperse the perfluoro compounds. Because systemic treatments require careful attention to physiological acceptability of the compounds, such as isotonic character, ionic . surfactants are less desirable, although it is possible . to offset or moderate their ionic character by formulating the dispersions with electrolytes or other additives.

Suitable nonionic sufactants include aliphatic materials such as block copolymers of ethylene oxide and propylene oxide comprising a hydrophobic propylene oxide section combined with one or more hydrophili« etylene oxide sections, for example the "Fluronic" (trademark) surfactants available from BASF Wyandotte, i4

» , 26149

Inc. Less desirably, aromatic types may also be used, i such as alkylphenoxypolyethoxyethanols having alkyl groups of about 7 to 18 carbon atoms and 1 to 60 or more oxyethyvlene units, for examples heptylphenory- polyethoryethanois, octylphenoxypolethoxyethanols, . methyloctylphenoxypolethoxyethanols, nonylphenoxypoly- ethoryethanols, dodecylphenoxypolyethoryethanols and the like: polyethoxyethanol derivatives of methylene linted alkylphenols; sul fur~containing analogs of the foregoing; ethylene oxide derivatives of long-chain carboxylic acids, such as lauric, myristic, palmitic, oleic, and the like or mivtures of acids such as are found in tall oil containing 1 to 60 ouyethylene units per molecules and analogous ethylene oxide condensates of long-chain or branched-chain amines, such as dodecylamine, hertadecyl amine, and octadecylamine, containing 1 to 60 ouyethylene groups.

Naturally occurring emulsifiers or derivaties thereof are also useful. These include the alginates, cellulose derivatives such as methyl cellulose and carboxymethyl cellulose, water soluble gums such as gum arabic and gum tragacanth, the phospholipids (such as lecithin and yolk phospholipid), and the sterols.

Nonionic fluorine containing surfactants are 29 particularly preferred. The fluorinated alkyl esters are one class of these surfactants, and are commercially available from 3M Company under. the designations FC-93, FC-95, FC-128, FC-143, FC-430 and

FC-431.

» . ° oq

The more preferred nonionic, Flaming containing © surfactanla, from the standpoint of Their exceptional ability ho Form dispersions which maiobsin a vange oy small parkicle size over schasbanbial poviorts rf Lime, orf Fhe order of 35 weeba bo oa wear or mesg es, £3 EY at room temperalurs, ars the (loorinalad amidoam ine oicles . cleacribed in WU.5. Fat. Wem. HBP, 0805 to Feice et al, and RAHAT ,99% Lo Parhlebb, the disc) msores of which are incorporated herein by referance, These compounds may 1n be generically described hy the formula LY : R CON—RQ

I

Y wherein R f is a perf Luoro a Phyl radical of 4 bo abowb 25 carbon atoms or a polyflaoroalloryall vl radical wherein the albkomiy group may contain 3 to aboul 40 car hon atoms of which at least a maior portion thereof Ate perflunrinated and the allyl group may con bain 2 bo about 40 carbon atoms, fluorinated or unfloorinateds VY is hydrogen or allyl of 1 to & car bran atoms; Rois an 2" alkylene radical of the foraulas —C Hy, — wherein = im oan integer of 1 to 6; and 0 is an ali phat coamine oxide radical of he formalas is —N —Rg } 3n 1 és :

» é& — wherein KR ant | are pach alkyl radicals of 1 to # : bi carbon atoms oF hydeosy-tecminabed alley 1 radicals of @

Lo & carbon atoms. In all cases She alloy, allyl and . alkylens groups may he straight or branched ahain,

Preferred subclasses of the surfactan La of I'he foregoing patents are those of bie following formulas (2) and (Rds

Q c F, +1(CF,) LN ) _NRIR® 1n n 2n 27x 2 BR wherein nis ab least 3 (prefecably A100, 3 is at

Ilmast 7 (preferably 206), y is Aa bolmast 1 {(prefecably 1 “

Reba) and Rand © independently are al Lod, vaclicals containing 1-6 carbon atoms, 0 i 1R2 i

C Fo, +1CNH(CH, ) NRIR 2n wherein © is at least 1 (preferably A100, = is at 1 2 least 1 (preferably 2-46), and Rand BB independently ars allyl radicals containing 1-6 carbon atoms.

Specific amidoaming oxi des within the scope of the shove formula are tha products described in Examples i 25 {fb of UJ.9. Fat. No. 3,928,085, name Tye

Q

CF_{(CF,) CNH(CH I. i 1

CF CFO(CF_) _CNH(C N(CH 3m (OF) JCFO(CF) CNH(CH, ) N(CH), 17 ro dD

BAD ORIGINAL £5 ’ Looe

0

I

(cr) erolcr,) nen, ) on, ), 26 1 4 A if 0 i (cr) Groce) func) on), ’ 0 i 1 (CF) CFO(CF,) CNH(CH,) N(CH, ),, 0 1n (CF) CFO(CF,)g (CH, ) | CNR(CH, ) JN(C, ig ),

The aqueous dispersions of the dovention are prepared by any mixing Lechnique which will provide a 2" uniform hlend of the ingredients, and peeparation accordingly may be readily accomplished by the sbilled formal ator. : oC When formulating the diepersions of the invenlion for systemic administration, it ods impor tan t rot anly ter add electrolytes and olher mat=rials bo renders Lhe dispersions prhyeioloyicst Ly moceptable (=uh ae imotonic with mammalian cells), Iveet alamo be adiuat Lhe pH, as necessary, to offset the lowering of the phoof

Fhe hypouic asl esd eanmen lt clos Fo generation of 3n lactic acid by Ll eo rypeveio bomen cml ls, MN mud table pl range is 7.97.4. Among the addi Lives commonly used bo

» ' © 26149 eC render {fluids physiologically acceptable are buffers . such as sodium bicarbonate, and mixtures such a

Ringer's Solution. Other materials conventionally employed in pharmaceutical preparations and known ta the skilled Formulator may also be added to the dispersions. These include viscasity modi fiers, stabilizers (against degradation due to freezing or contamination, for example), anti-freeze agents, } diluents, encoding agents, and the like. Among such additives may be mentioned glycerin, dimethyl-sulfoxide ("DMSO"), various gelatins both natural and synthetic, and polyols such as sorbitol.

The perfluoro compound and surfactant components may be blended into water in any proportions which will provide uniform dispersions. Typical proportions are about 5 to 50% perfluoro compound based on the volume " of the total composition and about 0.5 to 10% of the surfactant based on the total weight of the composition. Preferred proportions are about 10-30% by volume of the perfluoro compound and about 2-5% by . weight of the sw factant, but proportions in particular cases may be varied depending on disperability of the

FFC, particle size desired, and similar considerations.

The aqueous dispersions more usually comprise emulsions, preferably of the oil-in-water type but also including water-in-oil emulsions. In some cases the emulsions have a very fine particle size and appear transparent or solution-like to the unaided eye. The microemulsions which can be formulated with the

- , 26149 dispersants of U.S. Fat. No. 3,828,085 have this characteristic and are preferred. Colloidal suspensions, while not excluded from use in this invention, are less preferred, particularly for systemic administration, hecause of their larger particle size range and less stability. The above- identified blood substitute and surfactant patents provide eucellent guidance to formulation of the dispersions, and attention is directed to the patents for such purpose.

Sensitizing Treatment

The aqueous dispersions containing the oxygen transporting perfluoro compounds, when used as sensitizers in accordance with the invention, may be administered to a mammal locally or in any systemic fashion whether intravenous, subcutaneous, intramuscular, parenteral, intraperitoneal or oral.

Preferably, administration will be systemic and at a site enabling the dispersion to traverse the lungs to pick up oxygen and to transport the oxygen to the : hypoxic tumor cells. Dosages of the dispersion will be predetermined in accordance with the site and charcter of the hypoxia, whether or not the treatment is a supplement to hyperbaric oxygen treatment, the systemic tolerance (touicity) of the mammal to the specific formulation, and other factors known to the therapist.

Generally, fluorocrits (cc of PFC per 100 ml blood) of the perfluoro compound should be in the range of about

. 26149

A-10%, although Lower or higher fluorocrits in special circumstances may he sufficient or required. I+ the administration is a supplement bo hyperbaric oxygen treatment, ar other form of primary oxygen infusion, the fluorcrit need not be over 3.95%, and the partial pressure of oxygen in the inspired air may be up to about 2 atmospheres at 100% oxygen. As a maximum in most cases, hyperbaric oxygen administration would be 20 minutes at 2 atmospheres 100% oxygen pressure, and these conditions are known to be well within tolerated levels. However, the duration, content and pressure of the primary oxygenation in specific cases again will depend upon various factors, such as the health of the mammal or patient, the site of the hypoxia, and other conditions familiar to the radio- or chemotherapist.

Contact of the FFE dispersion may be with the hypoxic cells or with the tumor cell vasculature, such that the oxygen carried by the FFC may transfer to Lhe tumor /vascul ature interface. In other words, while the ideal may be direct contact hetween the FFO dispersion and the hypoxic cells, this may not be achievable and in fact is not required, since excess oxygen, wher =ver present in the tumor mass, will tend to become distributed throughout the mass, and thus reach the hypoxic cells.

The dosage of the sensitizing agent prior to irradiation and/or chemotherapy will also be controlled hy various conditions, including the rate at which the par fluora compound travels to the hypoxic tumor malls,

Lhe degree of sensitization desired, and the 2a

“ 20149 = cardiovascular hald4—1life residence time of bhe dispersion in the cardio-vascular system and in the hyporic tissues. For some treatments (such as hrief radiation treatments) an acceptable cardiovascular (serum) half-life can be as brief as about 2 to 4 hours. This duration indicates that the per-fluoro compound moves rapidly bo the hypendin Lumar cells and transfers its oxygen to the cells. In this connection an outstanding property of the preferred dispersions of the invention is an extremely small particle size, which particle size is maintained over substantial periods. The amall particle size enables the dispersions to quickly traverse the vasculature to the site of the hypoxia. For swample, an average particle size of 0.0% top 0.2 micron has been observed and has heen maintained for several months and up to a year or more .

The dispersions may be orygenated prior to infusion into the mammalian body and this may be expedient when injection is at or near the mite of the . hypoxia rather than at a site where oxygen transfer from the lungs and arteries is anticipated. rior } orygenation in auch manner may he accomplished by any means, . such as flushing or blanketing a eG ee containing the dispersion wilkh oxygen or air, or bubbling oxygen or air through the dispersion prior bo administration. When the treatment is a supplement to hyperbaric oxygen treatment, preamoygenation in the manner described may also be practiced In every CAs

DE i of preosygenabion, however, there may be a loss of odygen prior to entry of the dispersion into the region af the hypoxic tumor cells, that is, during transit of the dispersion to the oellsg hence, preorygenation generally is not preferred.

The types, mode of application and sites of radiation treatments aro well known and do not require detailed description. However, it will be evident that irradiation can be accomplished by external application

LO or by internal placement of radiation sources neal or at the site of the hyposi a. Accordingly, the irradiation may be achieved with x-rays, gamma ©FAye, neutrons and the like, or with implanted radium, iridium or cesium Sources. Conventional radiation therapy (200 rads per day, five days per week for sin ter eight weeks) may he employed hut dosage and total duration of treatment may be adjusted as required in particular circumstances.

The sensitizing method of the invention will he effective for all types of hypouic tumor cells, whether such cells be in suspension (as in leukemia) or in solid form, but the invention is particularly effective for anlid tumors. Recause systemic distribution of the dispersions is rapid, primarily due to the eutremsly zmall and stable particle size of the preaferrad dispersions of the invention, hypoxia at practically any site may be sensitized in accordance with the invention.

Chemotherapy is often used in combination with radiotherapy to destroy or control hypeostic tumor cells

* ’ " 26149 and therefore the sensitization techniques of the invention An he applied simul baneously or saquentially to chemobherapy and radiotherapy. hen dual therapy is used, a mensmi tice dieapersion will normally be selected vhich has the cardiovascular residence time effective to coc2r the dura tion of hokh treatments, or if the residence Fime is short, the sensitizer dosage can he suitably increased or adjusted. It is Erown that ME chemotherapeutic agents are oxygen dependent in terms of Fearing oxygen for active transport of the CT drug into the cell, For cell cycling control or CT enhancemsant.

Hence, oxygen must be supplied in free form or hy means of a carrier. Because the perfluoro compounds of the present invention and their dispersions are capable of transferring large quantities of oxygen, it wan be srpected that chemotherapy based upon drugs which are oxygen dependent will be benefited by formulating the drugs with ann RE agent of the present invention, ov sequentially administering the RS and CT agents. ’ Methotrexate is an example of a CT drug thought to reguire oxygen for active transport into the cell.

Vinhlastine and Vincristine are drugs which requice oxygen for cell cycling.

CT drugs which may not be oxygen dependent may also be administered in conjunction with the sensitizing techniques of the invention. Among such drugs may be mentioned Androgens, Estrogens, Anti mubtrogan, Frogesting, Achr-enal Steroids, IH br oogen =a

. 26149 9 .

Mustard, Chlorambucil, Fhenyl al anine Fash ard,

Cyclophosphamide, Thi o-TEFA, Busul fan, br

Mercaptopurine, H-Thioguanine, G-Fluorouracile,

Cytosine Ar-abineside, Adriamycin, Dactinoamyocin, 3 Daunomyecin, Rleomycin, Mithrameycin, Mitomycin-C, BON,

CNL, Methyl -CCMU, DTIC, Hydroxyarea, Cis-Flatinum (cis-platinum (ID) diamminedichloride), Frocarhazine,

Hex amethylmel amine, L-Asparaginase, and the like.

Associated Traatments

Those perfluoro compounds useful as RS agents but which also have radiopaque properties are particularly valuable for the purposes ofthe present invention.

Such compounds include brominated perfluorohydraocarbons such AB Feperfluoractylbromnide and brominated perfluoroethers, such as F—1 bramobutylisopropylether,

F-1, hbronoethylisopropyl ether and other brominated perfluoro organi ethers described, for example, in U.S.

Fat. No. 3,453,333. The radioimaging properties of such compounds permit monitoring of their RE effects as well as toxicity to surrounding normal cells and hence serve as diagnostic agents as well as RS agents.

However, if the perfluoro compounds do not also enthibit radiopacity, the dispersions containing the perf luoro ; compounts may be formulated with other, krown, radiopaque agents in order to provide a similar opportuni ty for monitoring radinsensitization potential. The radioimaging may be practiced as in conventional radiography or computer ad al tomoagranhy : (CATY radiography, or by the newer MHI bachii uns, The

. ’ . ' 26149 yo brominated compounds as RD agents may he used neat or in aqueous dispersion, for example as oil-in-water or water-in-oil emulaions containing about 10-F0% by valume of water and about 0.%5-10% by weight of a dispersant. fFladioprotection may also by £2 practiced in con junchion wi th the radiosensitization of the invention. Radioprotective agents are those which preferentially protect ner-mal tissues from eadiabtion injury. When practiced with radiosensitization, the objective ie to reduce injury to the normal Lissues, which injury may occur when the RS agents are used in the absence of the RF agents. Sul fhydryl containing agents generally are known Lo be effective FF materials, such as aminoethylisobthiwoniam or the phosphorotioate derivabives of hetamercaptosthylamine reviewed in the article by J. Il. Yutvas, "On the

Fotential Application of Radioprotective Drugs in Solid

Tumor Radiotherapy,” appearing in Radiation-Drug

Interactions in the Treatment of Cancer, edited by G6.

H. Sokal and . Fo. Haickel, Joba Wylie and Sons, Inc. (1980, pages 113-1035. Another RP agent is H-2- Ce aminopropylamingd ethylphosphorothioic acid, also described in the literature as WR-27T3L, This material provides protection hati in radiotherapy and chemotherapy, as described in the article by Yuhas et al appearing in Cancer Clinical Trials (1980), 3, 211- 216.

JS

" 260149

The following coeaples od 1] mere as fot hen i usb aki on of the dnvontion Yomibhenst, neceszmacri Ly

Timiting rhe scape theroof. hi be bhe seperiasnbal rasults of Euamples 0, A rnd CF wee abibained dn ei bee

Fhe sensitization demonsheabed by bhese ewaop Term im nok restricted to ponemamns lian systems beecanse the HITS studies are known ho correlate with op ab borne of the response of the cells io manma Toe Specifically, them

MTS are lnows bo esspood siad lacly bo the same Tome in when groan in the mammal Lan bor. :

EX aM 1 .

Preparation of PFO dispersions: (A) A moe factant Goll ion was prepared ky y dispersing in water a sufficient amount of Lhe

Tollowing amidpaming oxide surfactant CrOannT) to provide a 2% hy weight so Tukicn: 1 2A (Cry ),CFO(CF, )sCNH(CH, )3N(CHy )o ter ble sonication chamber of a "Sonic ator Chr adem) miving revice (Meat-Syshtens PIL Lm senna 05, Trev. , Plode] amy was added 2T co of the 2% sur fac tant malobion, followed by slow addition Lower Ted miehes) omvder Low moni cation power af ooo Cheogioee awk, Een 10 vol, . af a FFD composition consiabivg ef an about O/T by wend abt Tirpudel mites of Bob 3 Aida thy adamant ane ond

Feb d mest bg Vivi ope Tal TR nse, tev greene icle a Detaled

BAL ORIGINAL OF; i wae?

A 1 26149 30 ce of composition. (The FFE hivture was previously saturated with CO toy inhibit formation of fluoride ions). The sonication horn was then burned up to full power (setting of 10 on a scale of 1-10) for fone 35 minute followsd by a cooling period, This cycle was repeated about 1% times or until such time as 0» transparent, uniform dispersion was obtained. The dispersion (hereinafter identified as "Dispersion AY) was then filtered through a 0.22 micron "Millipore" 0 (trademark) filter and kept refrigerated at 4 (. until ,

USE. (RB) A second dispersion WAS prepared substantially as in (MN) above except for use of a nonionic polyoryethylene-polyorypropyl ene copol ymer surfactant having a molecular weight of about 8200 ("Pluronic" F-68) in place of the OND surfactant. This dispersion is hereinafter identified as "Dispersion BR." oc) A third dispersion was prepared substantially as described in (A) above except that the FFC was Fo tributyl amine (FC-43," 3M Company) and the surfactant } wag the "Fluronic F-69" of (RB). The proportions were the same (10 vol. % FEC, Bowl. % osu factant) This dispersion is hereinafter identified as "Dispersion cat ; EXareLE @

In Vitro Radiosensibtization

Multicellular tumor spheroids (CHTS™Y are produced

Fa) hy placing 10 Mita LL murine mammary bomen cell oan 10 mis of Eagles asal [esti vm CHET Y 0 Grand Te and

Riotogical Co., cabalag Mo, AE 1200, into a 100 mm . ae

‘ 26149 petri dish which has bean base coated with O.F5% noble

Rar in the EBREME. Within 7-10 days spherical agaregates of tumor cells appear and are then ready for study. The PFC dispersion to he tested is adjusted ho 2R0 milliosmoles osmolarity with powdered tissue culture medium and adivsted to pH 7.2 to T.4 with O01N

Mal or HEL. Other details of preparation and use of the MTS are given in J. il. Yuhas et al, 1977, "fy

Simplified Method for Prodoaction and Growth Of

Mul ticel lular Tumor Spheroids,” Cancer Research 3703639

AAA: and J. HM. Yuhas st al, 1978, "In Vitro Analysis

Of the Response OF Folticellular Tumor Spheroids

Exposed to Chemotherapeutic Agents In Vitro Or In

Vivo," Cancer Research 38: 3EG5- 09H.

Either standard tissue culture medium (EBHEY or the FFE dispersion is gassed with 100% oxygen for a period of fifteen miontes and then transferred, along with the spheroids, into non-heparinized capillary tubes and sealed. At intervals of © to 60 minutes later the tubes are exposed to graded doses of 250 kVp : X-rays, and within 30 minutes thereafter are removed, washed with medium and placed indidivaally in agar coated 16 mm wells along with 1.5 ce of medium. Using a dissecting scope at AQ power the spheroids are sized 2 three times weekly and the medium is changed twice weekly. From the growth curves for each group ia raleul ated the number of days required for pach spheraid to grow 200 or 150 om (niceons) in oi ameter larger than the size ab the beginning of the ne

, 260149 irradiation. The retardalion of rate of arowkh of the spheroids shown by the data therefore provides a measure of RS response where the Larger the difference over the irradiated controls the greater the RS effect, oS Tovicity of the FEC ie evaluated in terms of gerowbh delay, i.e., the retardation in growth of the spheroids after BHP OGLE ( the PIC Curd bhieat Prachi at ion relative to that obsevcsod in untreated spheroid oes,

Table 1 below shows the results of euperiments with

Dispersions A and B and Table Tl shows resulbts with

Dispersion OC.

It will be noted that Dispersions A and B provided similar levels of toxicity and radiosensitivity. The toxicity exhibited by Dispersions 0 and Bis considered negligible and likely to be relatively tolerable in the cardio-vascular system. The toxicity exhibited hy

Dispersion C, although higher than that of Dispersions

Nn and B, does nob disqualify Dispersion C from use as a sensitizer in mammal si such toxicity is relative to various factors, such as RE reagent and radiation dosage, and tumor Lype and location, and therefore

Dispersion CG would not necessarily be enrcluded from clinical evaluation. The data of Tables I and I1 is . not directly comparable: larger spheroids were aed in the euperiments reported in Table 1 than were used in the euperiments reported in Table 11. However, the larger spheroids provide added bias (larger spheroids are more difficult to sensitize) and therefore the resul te of Table I indicate highly beneficial

RO sensitization.

" ‘ 26149 e

On face value, it would appear that Dispersion C is a more effective radiosensitizer than either

Dispersion A or Dispersion B. This is in all probability not true for two reasons. The studies with

S Dispersion C were preliminary and involved MTE with a smaller fraction of radioresistant hypoxic cells at the time of treatment than was the case for the studies with Dispersions A and B. Second, Dispersion C was, in itself, growth inhibitory, and it is likely that this property in some way enhanced the tumor cell destruction.

TARLE I

TTT TTT TTT TT TTT TT TT TT Growth belay " Days to — (Days) » Radiation Grow Toxi- Radiation RS

System (rads) 200 pam city Effect Effect

MTS/EBME O 4.1 08 - -

MTS/EBME THO 5.73 ob 1.2°¢ -

MTS/EEME/ 0 4.4 0. =a —- -

Dispersion A

MTS/ERME/ THO P.O 0.30 4,464C 2.7

Dispersion A

MTS/EBME/ 0 4.1 nd - -

Dispersion B b -

MTS /EBME TSO 8.9 0 4.8¢ 3.6

Dispersion B

TARLE TI

TTT TT TTT TT TT TTT TT Growth Delay 23 Days to — Days) v Radiation Grow Toui- Radiation RS

System (rads) 150 pm city Effect Effect

MTS/EBME 0 3.8 0a - ws

MTS /EBME THO 11.5 ob 7.7 C -

MTS/EBME / 0 8.4 4.64 - -

Dispersion C

MTS/ERME/ THO 30 4.460 21.6 °C 18.5

Dispersion C :

BY definition.

Assumed to be same as unirradiated control. qOpserved time minus time required by respective unirradiated group.

Observed time minus time required by unirradiated control group.

. ' 26149 { . -

EXAMPLE 3

In Vivo Studies (Residence Times)

The rate of clearance of FFU dispersions A and EB from the circulaton was evaluated in both Fisher 344 } rats and BRALR/c mice (females in both cases). The dispersions were injected intravenously to a FFC doses equal to one-third of the circulatory volume, which is equal to 6% of body weight. Within 30 minutes of injection, the animal returns to the naormovolemic state as evidenced by the fact that the fluorocrit eguals 3.1% compared to the theoretical estimate of 3.3% for these Dispersions. At graded intervals through 8 hours after injection, blood is drawn in microcapillary tubes from the supraorbital sinus. Following centrifugation for 15 minutes at 12,000 times gravity, the fluorocrit (FFC as a percent of the blood volume) and the hematocrit are read on a microscope. The FFL collects i as a pellet in the bottom of the tubes, followed by the red blood cells and the plasma, thus forming distinct . layers. All data are normalized to the 30 minute centrifugation reading and the rate of decline of the fluorocrit is estimated from a standard single compartment exponential decay curve defined by: % PFC remaining = e~kt and i= 102 k

The results when plotted show that Dispersion A is superior to Dispersion B (half lives of Ab6H minutes and 222 minutes, respectively), probably because Dispersion

A has a smaller particle size than Dispersion RB. The . S results for the rats and mice were essentially equivalent. The results indicate that the FFC in both dispersions will clear the cardiovascular system quickly, and therefore both dispersions are good candidates for clinical studies.

EXAMPLE 4

The MTS experiments of Example 2? were repeated in all essential respects with Dispersion B but using spheroids derived from human tumor lines which contain hypoxic cells when grown as MTS. As shown in Table 111 below, some toxicity was observed in all cases but the levels are considered relatively tolerable. Mader ate radiosensitizing is apparent for the two neuroblastoma cell lines but very high radiosensitization is shown with respect to the melanoma line. The latter is an outstanding result due to the prevalence and high risks known for this form of hypoxic tumor cells. Very . little RS effect is shown for the osteosarcoma cell line but these results are only preliminary and the 29 exposure was at a low radiation level. . IR

’ 26149

TORLE 111 ersten ene

Days ta (Days)

Human Radiation (Fr-ow Toxi- Radiation RS

Tumor Line System (rads) 200 Hm city Effect Effect

NB-10 0 Neuroblastoma EEME E mre To TTT Te TTT TT re

EBME/MTH AO0 TL. 0 0.9 -

EEME/MTS/Dis. RB OQ bob 0. - -

EBME/MTS/Dis. B 400 0.10 0.1 x.4 2.6

LAN-1 Neuroblastoma ERME/MTS 0 a4, 0 - -

EBME/MTS 400 5.2 0 0.3 -

EBME/MTS/Dis. RB 0 Sa 0.3 - -

ERME/MTS/Dis. B 400 ?.3 0.3 4.1 4.1

SAQS Osteosarcoma EBME/MTS ) Bl. 6 0 = -

EBME/MTS 250 12.9 O 4.3 -

EBME/MTS/Dias. BR 0 10.7 2.1 - -

EBME/MTS/Dis. RB 250 14.0 2.1 3.3 1.1

C-32 Melanoma EBME/MTS 0 9 O bl - 1~ EBME/MTS THO 10.5 0 2.6 -

ERME/MTS/Dis. RH 8] 3.4 0.5 - -

ERME/MTS/Dis. B 7950 17.4 0.5 FO 6.9

EXAMPLE 5

Acute Toxicity

Studies were undertaken to confirm suitability of

FFL emulsions for intravascular administration and oxygen transport. The emulsion tested was Dispersion RB o which was held at 4 C. until use. Just prior to use 2n the osmolarity was adjusted to approximately 300 milliosmoles with powdered tissue culture medium ’ (Eagles Basal Medium-"EBME"), and the pH adjusted to 7.4 with HCL or NaOH. Table IV below summarizes the results of an acute toxicity study on Fisher - 344 rats in which various doses of the emulsion were injected into the tail veins of the rats. Rats reported as surviving after injection survived for at least ten days. Deaths normally occurred in 4 to 24 hours if at all. The results indicate ‘that the animals can xa

- ‘ tolerate large doses of PFC emulsion relative to their blood volume (about &0 ta 80 ml/kg) as compared with the doses which would be administered to assess radiosensitization in vivo, namely, 20 to 730 ml/kg. In .

LD (I.V.) tests it was determined that the lethal 50 dosage was greater than 60 ml/kg for BALB/c mice and greater than 120 ml/kg for the Fisher - 344 rats. The observations are consistent with those reported by other investigators in that the FFC dispersion has a low acute toxicity even when administered hypervolemically at doses exceeding the animal ’s normal hypervolemically blood volume. The deaths which occurred at the high doses probably were due to fluid overload rather than inherent toxicity of the FFC dispersions.

TARLE IV

~TTTTTTTTAGUTE TOXICITY OF DISPERSION B

ADMINISTERED HYFERVOLEMICALLY 1.V. IN RATS

NUMBER DEAD + } DOSE (ml/kg) NUMBER INJECTED 20 a/11 40 0/3 ’ HO Qa/73 80 0/3 100 0/28 . 120 1/773 80ne animal died during injection due to mishandling.

EXAMPLE & .

In Vivo Radiosensitization

Radiosensitization of the 3MIN mammary tumor growing in the right hind leg of Fisher — 344 rats was studied. x3

» ! 26149

At 10-14 days after subcutaneous transplantation in the rats the tumors were 6-8 om in diameter and ready for treatment. The rontrol animals either received no treatment or received various radiation doses. The

S other animals received 1.V. infusion of 20 ml/kg of

Dispersion B (20% w/v) followed by 30 minutes breathing of 95% 0 /5% CO gas mixture (at 1 atm) and then by ~ > the various radiation doses of u-rays. Frior to and three times weekly after treatment, the two orthogonal diameters of the tumors were measured in situ and averaged. Table V below expresses the results of the study as the time required for the tumors to grow 8 mm beyond their size at the time of treatment. The data show that the enhancement oroduced hy the FFC dispersion increases with radiation dose, and also that the growth delay per rat is significantly higher in the

FFC~treated group than in the control. That the enhancement is due to the combination of the FC dispersion and breathing of the gas mixture is evident from Table VI below in that none of the control . . treatments were capable of producing the growth delay of the combination. 29 3b q " 49 0

TARE V re TEENS TT IZATION OF SMM TUMORS IN RATS

AT DIFFERENT RADIATION Co

POSES WITH DISFERSION R

DAYS TO GROW TO 8 mm 9 eestor emer

RADIATION DIGFERSION B

DOSE (RADS) CONTROLS FLUS HIGH OXYGEN 0 11.5 + 0.8 11.5 4 1.1

S500 10.9 + 0.9 12.0 £ 0.9 1500 19.5 + 0.9 24.9 * 0.8

PHOO 25.2 % 1.4 5.7 + 1.3

TABLE VI

EE ETE OF VARIOUS CONTROL TREATMENTS

ON GROWTH OF 3M2N TUMORS IN RATS

Mone 12.3 + 1.4 -

Emulsion (8) 12.9 * 0.9 0.6 2,500 Rags RT. ok 2.4 15.0

Saline \&/+ 25.3 * 3.1 13.0 2,500 Ras

Emul sion a), 24.4 + 2.8 12.1 2,500 Rads 95% CO /5% CO, + 29.8 + 3.1 17.95 2 2 2,500 Rads (2)20 m1/x ml/kg of Dispersion B or equal volume dose of saline. :

EXAMPLE 7

In Vitro Chemosensitization

The potentiation by FFC dispersions of chemotherapeutic agents was demonstrated by the anti- tumor effect of methotrexate ("MTX") in NR-—1 G0 neuroblastoma multicellular tumor spheroids ("MTS").

In this study, the speroids were suposed to from 0 to ~é

Se LO mol ar methotrerate in control medium and in

IF

Dispersion B equilibrated with a 95% 0 /5% CO mixture. 2 2

The results (Table VII below) show that the FFC dispersion enhanced the effectiveness of MTX as evidenced by the delay in growth relative to the spheroids treated only with MTX. Al though some sensitization to MTX in these spheroids can be achieved by gassing with the gas mixture in medium alone, such sensitization requires 1 to 2 hour pretreatment as opposed to only 30 minutes with the FFC dispersion, thus clearly demonstrating the feasibility of elevating the therapeautic index of this important anti-cancer drug with PFC.

TABLE VII

Effects of FFC Dispersion BE and oxygen on the response of

NE-100 MTS to a 24 hour exposure of J uM MIX for 24 hours

FFG

CONTROLS MTX DISFERSTON 4+ MTX ee ok ym

Day 0 2.7 = 0.31 9.83% &£ 0,17 9.90 + 0.23 1. 10.23 ?.74 PTT 4 13.78 10.6% ?. 66 6 15.72 11.76 2.83 a 17.17 13.93 10.90 12 18.93 15.81 15 21.63 19.0 : 18 23.5 pg P 7.18 7.52 13.02

MTS sizes are given in microscope units, where 1 unit = 25 um pg is the number of days required for the MTS to grow 8 units or 200 um beyond their original diameter. 29 EXAMPLE 8 . .

In Vivo Chemosensitization

The combined use of PFC dispersion (Dispersion RB) and high oxygen breathing for enhancement of the anti- aa

' tumor effects of cyclophosphamide ¢cyec™) Was investigated with respect to treatment of MCa-11 mammary carcinoma transplanted into the right hind leg of female BALB/c mice. The tumors were allowed to grow to 2 mm at which point the animals were treated as described in Table VIII below, wherein the cyclophosphamide, obtained commercially, was dissolved in distilled water and injected in a volume equal to 0.01 ml/kg of body weight. Three to six animals per treatment group were used in the study. On the day of : treatment and three to five times per week thereafter, the tumors were sized with vernier calipers.

From the test results (Table VIII), it is evident that the anti-tumor effect (growth delay) produced by the cyclophosphamide alone (absence of oxygen and/or

FFC dispersion) was not detectably enhanced by oxygen breathing (of 95% 0 /5% CO mixture) or by injection of

FFC dispersion followed by oxygen breathing, but was enhanced effectively by administration of FEC dispersion in concert with oxygen breathing.

TABLE VIII eee eee eens es creeeemetmemremcr Bes

Effects of FFC = Oxygen on the Antitumor }

Effectiveness of Cyclophosphamide

TTT Number Days to

Treatment @ of Mice Grow & mm.Y Growth Delay

Controls 14 12.5 -

Cyclophosphamide 5S 17.8 5.3 : FFC + CYC 4 17.1 4.5

Oxygen + CYC 5 1éab 4.1

FFC + Oxy. + CYC b 20.8 8.3 eee eer emrene et eee 8controls = no treatment; Cyclophosphamide = given a single dose of

T5 mg/kg of cyclophosphamide via i.p. injection; PFC = given a single injection of 20 ml/kg of F-DMA/F-NONAN 2 hours before receipt of the cyclophosphamide; and Oxygen = maintained in an atmosphere of 95% oxygen + 5% COofor 2 hours before and two hours after administration of the cyclophos- phemide. In the combined PFC + oxygen group, the PFC was administered just poefore the mice were placed in the 95% oxygen atmosphere. the days required to grow 6 mm were interpolated from the tumor growth curves constructed from the test data.

. i

EXAMPLE © 20 1 4 9 i

In Vivo Chemoprotection ahd Chemosensi tization

Combined chemosensitization of hypoxic tumors to chemotherapeutic agents and protection of normal tissues is achieved by the procedure described by Yuhas at al, "Treatment of Tumours with the Combination of

WR-2721 and Cis-dichlorodiammine platinum (In or

Cyclophosphamide,” Br. J. Cancer (198M, 42,574-585 and publications referenced therein, as modified by concurrent treatment with 20 ml/kg of a FFC dispersion, such as Dispersion B, as described in Fxample 8 above, to accommodate lower dosages of the chemo—-therapeutic agents (7Smg/kg), room air breathing, high oxygen (95% 0 /S% C0 ) breathing, controls, and other conditions 2 2 as appropriate.

EXAMPLE 10

In Vivo Radioprotection and Radiosensitization

The sensitization of hypoxic tumors to irradiation : by t*eatmant with FFC compositions of the invention in combination with radioprotection of normal tissues in the region of treatment is achieved by the procedure .- # described by Yuhas et al, "The Role of WR-2721 In

Radiotherapy and/or Chemotherapy," Cancer Clinical

Trials (1980), 3,211-216 and publications referenced therein, modified as described in Example é6 above to provide for concurrent infusion of 20 ml/kg of a FFC composition auch as Dispersion RB, high yen

- ’ 26149 (95% © /5% CO) or air breathing, controls, and -! other conditions as appropriate. : Delivery of Lipophilic Drugs

In the course of in vitro chemosensitization studies in the LAN-1 human neuroblastoma spheroid system, conducted substantially as described in Example 4 above except for Adriamycin as the chemotherapeutic . agent, it was noted that the anti-tumor activity of the

Adriamycin was inhibited by the FFC material (PFC

Dispersion BR). This suggested that the FFL may have baen physically binding the Adriamycin, thereby reducing the concentration available for absorption by the spheroids. This was tested by dissolving in water a radio-labelled analogue of Adriamycin, co paunomycin, adding Dispersion B (or in separate experiments, the neat PFC component of Dispersion BE), blending for 30 minutes, separating the FFC phase from the agueous phase, and determining the distribution of drug between the phases. Table IX below reports the results of the ] experiment and similar experiments with three other drugs. It will be noted that the more lipophilic

Daunomycin preferentially partitioned into the FC phase whereas the other, less lipophilic, drugs favored the aqueous phase. It was concluded that although the

PFC may have an inhibitory effect on less lipophilic drugs, the delivery of lipophilic drugs will be enhanced by the FFC.

: ) 49

TARLE IX 26 1

TTT BINDING OF FOUR CANCER TREATMENT

DRUGS RY FFC DISFERSION

Lipophilicity Drug in Emulsion +

Drug Indes Drug in Aqueous Fhase 3 FE ,

Daunomycin 0.79 3.77

Misonidazole 0.43 0.04

Methotrenate 0.014 0.07

WR—-2721 0.002 0.05 ¥Lipophilicity index is the partition coefficient of the drug between octanol and water.

The preferential solublity of lipophilic drugs in

FFC compositions not only provides a oeans far enhancing the delivery of such drugs in animals but also opens up opportunity for controlling residence time of the drug in the animal, for example by prolonging release of the drug from the FFC circulating to the plasma and target organs. In other words, by appropriate selection af a drug from the standpoint of its relative lipophilicity in a FFC dispersion, sustained low level delivery of a drug for a prolonged period is made possible with an agueous delivery system.

EXAMFLE 11 . Enhancement of Chemotherapy

This example illustrates enhancement of anti-tumor action of Vincristine, a lipophilic drug to which tumors are normally resistent at least to non-lethal doses thereof. The enhancement is believed due to the preferential solubility of the drug in the FFC material. !

=

The partitioning of the Vincristine between the

FFC and aqueous phases of Dispersion EB was first studied in comparison with Daunomycin, as reported in

Table X below, where "FFC" means the FFC phase of

Dispersion R.

TABLE X i, 6b

Octanol s Water ii,

Drug Fartition Coefficient 1 hour 3 hours

Paunomycin Tulse TTT er Bue

Vincristine 682 2.6% ?.10C & The higher the octanol:water partition coefficient, the more lipophilic b is the drug. ratio of concentration in the emulsion and the aqueous phases. determined via bioassay in the LAN-1 neuroblastoma spheroid system.

Thereafter, in vivo chemosensitization was - 15 conducted in MCa-11 mammary, carcinoma essentially as described in Example B above except as follows:

The MCa—-13i mammary carcinoma was transplanted into the thigh of BALB/c mice and grown to a diameter of 6 mm, at which time the mice were treated. Control mice received an injection of saline, while Vincristine ’ treatment consisted of a single dose of 1.5 mg/kg of

Vincristine administered intraperitoneally. FFC treatment consisted of an i.v. injection of 20 ml/kg of

Dispersion E, followed by two hours of breathing carbogen (95% 0 /5% CO ), and injection of saline or

Vincristine as before, followed by another two hours of carbogen breathing. It was observed that the

Vincristine treatment alone had no effect on tumor growth. Similarly, administration of the FFC emulsion

" | ’ 26149 alone had no effects on tumor growth, but when combined with the injection of Vincristine, the growth of the tumor was delayed by approximately $ days. Dv ygen breathing was included in the experiment in order to guarantee that any stimulation of the active efflux mechanism might not override any potential benefits gained via a more advantageous delivery of drug. In vitro studies now underway indicate that the benefits of the FFC-Vincristine combination are not oxygen dependent. Moreover, enhanced host toxicity of the

Vincristine when combined with the FFC dispersion could not be detected. Thus, it appears that FFC dispersions have the potential to deliver low levels of drugs over prolonged periods.

It will also be apparent that by matching FFC materials and drugs based on their relative lipophilicity and the rate of clearance of FFC material from the body, the efficacy of drugs in a variety of treatments may be controlled. In some cases the effect may be enhancement of activity. In other cases diminished activity or prolonged activity may be the preferred result. The latter effect is obtainable, for erample, by selection of a FFO carrier material which is known to clear more slowly from the body than will another PFC material. These benefits are achievable whether or not the FFC material is also being utilized as an oxygen transporting agent.

There are a number of instances in which the solubility of drugs in FFOC could be exploited in medicine. If, as in the cases mentioned above, one

’ ” at 26149 ° selects a highly lipophilic drug which preferentially partitions into the organic, FFC phase, it is possible to produce a sustained drug delivery system, As the tissues absorb the drug from the aqueous phase, a new equilibrium will be established, i.e., some of the drug in the organic phase will transfer back into the agueous phase. In practice, this would be a continuous process, which provides chronic, low level dr-ug eRpPasSure. Three general classes of drugs can be envisoned: those which are totally insoluble in FFC (very hydrophilic), those which are of similar solubility in the FFC and water, and those which are more suluble in the FFC (very lipophilic). While lipophilicity is not the sole determinant of solubility in the two phases, it is a major determinant and can he used as a guide in the selection of candidate drugs.

One additional characteristic must be considered, the "loading factor"; this is the absolute solubility of the drug in the FFC. The emulsion must be able to carry a therapeutic dose level, i.e., not become - saturated at less than desirable concentrations.

Specific applications of this approach include, but are not limited to: (1) chronic administration of lipophilic, phase specific cancer chemotherapeutics, such as Vincristine sulfate; (2) chronic administration of lipophilic cardiovascular agents, such as Digitoming (3) chronic administration of lipophilic nutritional supplements like Vitamin Ej (4) chronic administration of lipophilic hormones, such as Bstradiols and (35)

A

» RB 26149 chronic administration of any lipohilic agent whose effectiveness would be enhanced or whose toxicity would be reduced via chronic administration.

In those instances where water solubility is too low for effective dispersion in a FFC emulsion, the drug can be introduced directly into the FFC, followed by dispersion of the FFC in water.

Useful guidance to drug absorption is present in "Pradiction OF The Volume Of Distribution From In Vitro

Data And Use for Estimating The Ahsolute Extent OF

Absorption, W. A. Ritaschel and G. V. Hammer,

International Journal of Clinical Fharmacology, Therapy and Toxicology, 18:298-316 (No. Ty 1980). This article (incorporated herein hy reference) defines Apparent

Distribution Coefficient (AFD) in a huffered octanol/— water mixture as follows:

APC = (Cp - Cy ) .a

Co? where CO is drug concentration in the aqueous phase before equilibrium, 0a is drug concentration in aqueous phase after equilibrium, a is volume of aqueous phase, and b is volume of the octanol phase. The misvture is at equilibrium when the concentrations of drug in the octanol and aqueous phases remain constant at a given temperature after suitable agitation. In the system used to determine AFCs in the Ritschel and

. | ‘ ! 26149

Hammer article, equilibrium was reached after 8 hours agitation at 7G. AFC as defined above is useful in selecting drugs whose therapeutic efficacy can he enhanced pursuant to the present invention.

Freferably the drug is one having an AFC (in a 50/50 by volume equilibrium mixtuwe of water and the perfluorocarbon compound or mixture of perfluorocarbon . compounds) reflecting a level of absorption into the

FFC phase effective for therapeutic blood levels, such as at least 1.0, more preferably over 1.0. Equilibrium will be the point of admixture at which the concentration of drug in each of the two phases is constant at 7 oC. Buffering of the FFO dispersion is optional.

The foregoing describes some aspects of lipophilic drug manipulation for improved delivery and therapeutic effect. It will be apparent that by matching FFC compounds and drugs on the basis of their relative lipophilicity in aqueous media, it is now possible to determine the amount of a specific drug to be added to a perfluorocarbon dispersion in order to deliver a concentration of the drug in the blood in a therapeutic range (therapeutic concentration ranges are known for all approved drugs), since knowing the lipophilicity of 29 the drug in the FFC dispersion by AFC measurement, one has guidance to prediction of how much of the drug will he available for therapeutic effect and the rate at which the drug will be made available. 0M wide spectrum of therapeutic effect is, of course, represented by the great variety of lipophilic drugs available as

¥ 26149 . indicated, far example, in the current edition of The

Merck Index and similar compilations. The drugs thus include many chemotherapeutic agents, chemotherapeutic protective agents and radioprotective agents such as’ those identified herein, as well as the drugs of the

Ritschel and Hammer article cited above. The invention thus opens up vast opportunity for enhanced therapy over a wide spectrum of medical treatments.

AR

Claims (2)

. I" Vv , i : ! 26149 We claim: ed NJ

1. A method of producing a perfluorocarbon- containing aqueous emulsion containing an additional pharmaceutically useful material selected from the group consisting of lipophilic drugs, chemotherapeutic agents, chemotherapeutic protective agents and radioprotective agents which comprises the steps of: 1) admixing the perfluorocarbon with an aqueous solution of a dispersant selected from ethylene oxide/propylene oxide block copolymers, polyethoxylated alkyl phenols, polyethoxylated fatty alcohols, fluorinated alkyl esters and fluorinated amidoamine oxides to produce an aqueous composition comprising 5-50% perfluoro compound and 0.5-10% dispersant while subjecting the mixture to a low power dispersion technique to produce an emulsion of micelle size 0.5 to 1 micron, and 2) submitting the product to a high power dispersion technique to produce an emulsion of micelle size 0.05 to 0.2 micron, and 3) admixing said lipophilic drug, chemo- therapeutic agent, chemotherapeutic protective agent or radioprotective agent.

2. A method according to claim 1, wherein the perfluorocarbon is presaturated with carbon dioxide.

i ARSTRACT

Method of sensitizing hyporic tumor cells to radiotherapy and chemotherapy by contacting the cells or the vasculature thereof with an aqueous dispersion of an oxygen carrying perf uoro compound and a dispersant for the compound , and sensitizing chemotherapeutic and protective compositions therefor.