CA3175637A1 - Composition and method for oral treatment of leukemia - Google Patents

Abstract

A method of treating a mammalian subject having hematologic, non-tumorous cancer cells is disclosed. The method comprises the steps of: (A) administering to such a mammalian subject a therapeutically effective amount of a halogenated xanthene, a pharmaceutically acceptable salt or a C1-C4 alkyl ester thereof as a first cancer cytotoxic agent dissolved or dispersed in a pharmaceutically acceptable aqueous medium. The mammalian subject is maintained for a period of time sufficient to induce death of hematologic, non-tumorous cancer cells. A contemplated administration is typically repeated. A contemplated treatment method can also be carried out in conjunction with administration to said mammalian subject of a second therapeutically effective amount of a second, differently-acting cancer cytotoxic agent dissolved or dispersed in a pharmaceutically acceptable medium. The second cancer cytotoxic agent can be a small molecule or an intact antibody or paratope-containing portion thereof.

Description

COMPOSITION AND METHOD FOR
ORAL TREATMENT OF LEUKEMIA
Descri pti on Field of the Invention This invention relates to an oral therapeutic regi men for t reat i ng bl ood ( hematol ogi c) cancers such as leukemia particularly effecting such treatments i n chi I dren.
BACKGROUND ART
An adult human has about 7000 white bl ood cell s per mi crol i ter (IL) of bl ood. Of those whi te cells, about 65 percent are granul ocytes ( about 4500/ L), about 30 percent are monocyt es ( about 2100/ L), and about five percent are lymphocytes ( about 350/ L) . Geyt on, Text book of Medical Physiology, Seventh ed. , W. B. Saunders Co., Philadelphia ( 1986).
The above cell number amounts are, of course, general i zed average val ues, and granul ocyte counts for normal pat i ents, i . e. , pat i ents free of disease, typi call y are about 2000 to about 7000 cell s/ L.
Acute I ymphobl ast i c I eukemi a (ALL) i s a cancer of the I ymphoi d I i ne of bl ood cel I s begi nni ng i n the bone marrow, and characteri zed by the devel opment of large numbers of immature lymphocytes ( I ymphobl asts). There are two basic types of this di sease. One affects B cell s ( B- ALL) , and the other affects T cells ( T- ALL) . As an acute leukemia, ALL

progresses rapi dl y and i s typi cal I y fatal wi t hi n weeks or months if I eft untreated.
ALL occurs i n both chi I dren and adul ts, with highest rates seen between the ages three and seven years. About 75 percent of cases occur before the age of 6, with a secondary rise after the age of 40. The overall i nci dence of pedi at r i c ALL i n the Uni ted States dun i ng 2001-2014 was 34.0 cases per 1 mill i on persons and among all raci al /et hni c groups.
ALL i s typi cal I y treated i ni ti ally with chemotherapy aimed at bringing about remission.
This is then foil owed by further chemotherapy typically over three years. Treatment usual I y al so i ncl udes i nt rat hecal chemotherapy (spinal cord i nj ect i on), because systemi c chemotherapy can have limited penetration i nto the central nervous system and the central nervous system i s a common site for rel apse of ALL.
Chronic I ymphocyti c leukemia (CLL) is a type of cancer i n whi ch the bone marrow makes too many lymphocytes, part i cul an y B cel Is. Al though it is general I y consi dered i ncurabl e, CLL progresses sl owl y i n most cases. CLL treatment consequently focuses on control I i ng the di sease and its symptoms rather than on an outright cure. The decision to start CLL treatment is taken when the person' s symptoms or blood counts i ndi cate that the di sease has progressed to a poi nt where it may affect quality of life.
CLL i s pri man i I y a di sease of ol der adul ts, most commonly occur ri ng i n peopl e over the age of 50, with a median age of 70 years at the ti me of diagnosis.
Though I ess common, CLL someti mes affects peopl e

- 2 -between 30 and 39 years of age. The i nci dence of CLL
increases very qui ckl y with i ncreasi ng age.
Five-year survi val foil owi ng di agnosi s i s approxi mat el y 83% i n the United States.
Acute myel ogenous leukemia (AML) begins in the bone marrow as a di sorder of the hematopoi et i c stem cell and i s the most common form of I eukemi a i n adul ts.
It occurs i n both chi I dren and adults. Without treatment, AML can rapidly progress in the body as new white blood cells conti nue bei ng made.
Chronic myel ogenous leukemia (CML), al so known as chronic granulocytic leukemia (CGL), al so starts i n the bone marrow, but it progresses I ess rapidly than AML. In its early phases, CML is characterized by I eukocytosi s, the presence of i ncreased numbers of i mature granul ocytes i n the per i pheral bl ood, spl enomegal y and anemi a. These immature granul ocyt es i ncl ude basophi I s, eosi nophi Is, and neutrophi I s. The i mature granul ocyt es al so accumul ate i n the bone marrow, spl een, liver, and occasi onal ly in other ti ssues. Pat i ents present i ng with this disease characteri st i cal I y have more than 75, 000 white blood cells per mi crol i ter (jIL) , and the count can exceed 500, 000/ L.
CML accounts for about 20 percent of all I eukemi as i n the Uni ted States. About 15 new cases per mill i on peopl e are reported each year, I eadi ng to about

3,000 to 4,000 new cases per year. The disease is rare i n humans bel ow age 45, but i nci dence ri ses rapi dl y to age 65, and remai ns el evated thereafter. The medi an life span of pati ents with chronic myel ogenous leukemia from the ti me of diagnosis is approximately four years.
About 60 to 80 percent of pat i ents with CML
devel op a bl ast cri Si S.
BI ast cri Si s represents a mani f estati on of acute I eukemi a. The presence of certai n markers on the bl ast cell s someti mes suggests a I ymphoi d or i gi n of these cel I s dun i ng the bl ast cri Si S.
Chemotherapeuti c agents used for the treatment of the bl ast cri Si s are the same as those used for the treatment of other acute leukemias.
For exampl e, cytarabi ne and daunorubi ci n, used for the treatment of acute myel ocyti c I eukemi a, are used to treat CML bl ast cri si S.
Predni sone and vi ncri sti ne, a therapeuti c regi me used i n the treatment of acute I ymphocyti c leukemias, is al so used to treat CML bl ast crisis.
Nevertheless, these drug therapies of the bl ast cri Si s stage of CML are even I ess successful than are the treatments of other acute leukemias.
Cancer i n chi I dren i s rare wi th an i nci dence of 140- 155 per mill i on ( age <15 years) per year.
Thi s transl ates to about 1 i n 7,000 chi I dren is diagnosed with cancer each year.
Despite the rarity of cancer, mal i gnant neopl asm i s the most common cause of death after acci dents i n chi I dren aged 5 to 14 years, accounti ng for 23 percent of mortal i ty.
Survi val from chi I dhood cancers, many of whi ch were fatal i n the pre-chemotherapy era, has i ncreased dramatically from 20 to 30 percent i n the 1960s to 62 percent i n the 1970s, and more recently to 83 percent. Sal etta et al . , Trans!
Pedi at r 3(2): 156- 182 (2014).
Leukemi as are the most common chi I dhood cancers, accounti ng for about 30% of all pedi at ri c

- 4 -( ages 1-14) cancer diagnoses. Acute I ymphobl asti c 1 eukemi a (ALL) accounts for about 25 percent of chi 1 drens' cancers, and acute myel oi d I eukemi a (AML) accounts for the remai ni ng about 5 percent.
Sal et ta et al . , Trans! Pedi at r 3( 2) : 156-182 ( 2014) Current treatments for ALL i ncl ude pegyl at ed aspargi nase, I i posomal daunorubi ci n, I i posomal annamyci n, sphi ngosomal vi ncri st i ne, and 1 i posomal cytarabi ne. For AML, current treatments include the use of all t rans- ret i noi c acid (ATRA) , arsenic t ri oxi de, ant hracycl i ne combi ned with ATRA, and i darubi ci n with high-dose cytarabi ne. Soraf eni b (multi ki nase i nhi bi tor) i n combination with cl of arabi ne and cytarabi ne has found success i n a phase 1 study [
Inaba et al . , J CI i n Oncol 29: 3293-3300 ( 2011) ], and a cal i cheami ci n- conj ugated CD33 anti body, gemt uzumab ozogami ci n, known commerci al 1 y as Myl otarg , has shown promi se [ Zwaan et al . , Br J Haematol 148: 768-776 (2010)1.
Al though the survival rate for pediatric 1 eukemi a has great I y i mproved, rel apse i s a maj or cause of treatment fail ure. Approxi mate! y 15 to 20 percent of pediatric ALL patients and 30 to 40 percent of AML
pat i ents rel apse, with relapsed ALL i dent i f i ed as the fourth most common mal i gnancy i n children.
Treatment of relapsed pediatric 1 eukemi a i ncl udes i nt ensi f i cat i on of chemot her apeut i c regi mens and use of bone marrow transplantation ( BMT) .
However, i ncreasi ng the i nt ensi ty of combi nat i on chemot herapi es and introduction of second-1 i ne drugs is of ten accompani ed by cumulative toxi city, with margi nal i ncrement al benef i ts.

- 5 -A key component for understandi ng i mmune system i nteract i ons agai nst pediatric cancers is the avail ability of an appl i cabl e ani mal model .
Current xenograf t model s are I i mi ted because they are established i n severe combi ned i mmunodef i ci ent ( SCI D) mi ce and so do not provi de i nf ormati on on the contri but i on of the i mmune system.
Other approaches such as human hematopoi et i c stem cell reconsti t uti on i n i mmunocompetent animals are cumbersome, expensive, and of ten i nt roduce compl ex bi ol ogi cal variables i nto the syst em.
Recently, a novel xenograf t tumor model was devel oped i n i mmunocompet ent mi ce by t ol en i zi ng mi ce fetuses to human t umor cell s [ Basel et al . , Cancer Lett. 412: 256- 263 ( 2018) ] . This model is advantageous because it can be used to better describe the complex i nteracti on between cancerous cell s and the i mmune system through a xenograf t technique.
One useful anti-cancer agent group for adult cancerous tumors are the hal ogenated xanthenes, or the pharmaceutically acceptable salts thereof. See, US
Patents No. 6, 331, 286, No. 7, 390, 668, No. 7, 648, 695, No. 9, 107, 887, No. 9, 808, 524, No. 9, 839, 688, and No.
10, 130, 658. Of those hal ogenated xanthenes, Rose Bengal di sodi um, ( 4, 5, 6, 7- t et rachl or o- 2' , 4' , 5' , 7' -tet rai odof I uorescei n di sodi um; RB) has been found to be part i cul ar I y effective and easi I y utilized.
A sol ut i on of i odi ne- 131 radi ol abel ed RB has been used cl i ni cal I y to measure liver f unct i on i n infants [ Yvart et al . , Eur J Nucl Med 6:355- 359 ( 1981) ]. PV- ioe, a steri I e 10 percent w/v RB di sodi um

- 6 -sol ut i on i n aqueous 0.9% sodi um chi or i de for i nj ect i on is a more recent formulation that is manufactured by Provect us Bi opharmaceut i cal s, I nc. of Knoxvi I I e, TN.
Previ ous studi es have shown that RB or its salt from PV- 10 aqueous RB di sodi urn solution accumul at es i n cancerous cel I I ysosomes [Wachter, et al . , Proceedi ngs of SPI E, Multi photon Microscopy i n the Bi omedi cal Sci ences I I , Pen i asamy, A. and So, P. T. C.
( eds), Bell i ngham, Washi ngt on: 4620: 143-147 ( 2002) ]
and i nduces cell death in a range of adult cancers [Qi n et al . , Cel I Death Di s 8: e2584 ( 2017); Toomey et al PloS ONE 8( 7) : e68561 ( 2013); Koevary et al . , Intl Physi ol Pat hophysi ol Pharmacol 4( 2) : 99-107 ( 2012);
Thompson et al . , Mel anoma Res 18( 6) :405-411 ( 2008); and Zamani et al . , J I mmunotoxi c 11( 4) : 367-375 ( 2014) ] .
PV- 108 aqueous RB di sodi urn solution has been used i n several cl i ni cal t ri al s, both as a si ngl e anti -cancer agent and i n conj unct i on with monad l onal anti body anti -cancer agents, where it has been admi ni stered i nto solid tumor cancers via i nt ral esi onal ( I L) administration. Several of those trials are di scussed bel ow. Phase I and phase I I cl i ni cal st udi es usi ng PV- 10 aqueous RB di sodi urn sol uti on al one as the cyt ot oxi c agent ill ust rat i vel y reported "adverse events were predomi nantl y mi I d to moderate and I ocoregi onal to the treatment site, with no treatment-associated grade 4 or 5 adverse events" [ Thompson et al . , Ann Surg Oncol 22( 7) : 2135-2142 ( 2015) ] , and "Tr eat ment - Emer gent Adverse Events (TEAEs) were consi stent with est abl i shed patterns for each drug, pr i nci pal I y Grade 1-2 i nj ect i on site react i ons at t ri but ed to PV- 108 aqueous RB di sodi urn

- 7 -solution and Grade 1-3 immune-mediated reactions att ri buted to pembrol i zumab, with no si gni f i cant over I ap or unexpected toxi ci ti es: . . . " [ Agarwal a et al . , J CH n Oncol 3]( 15) suppl 9559-9559 (May 26, 2019) ] . It thus appears as though RB is toxic to cancerous cell s, but non- toxi c to non- cancerous cell s.
Because of the often-times very different behavior of adult tumors from pediatric tumors, it was not known whether RB and si mi I ar hal ogenated xanthenes woul d be effective when used agai nst pediatric cancerous cells and, particularly, pedi at ri c cancerous hematol ogi c cells.
Prel i mi nary in vitro and xenograf t st udi es agai nst neurobl astoma cell I i nes i n cell cul tures to whi ch RB was added al one or i n conj uncti on with known anti cancer agents, and by i ntral esi onal injection i nto sol i d tumor xenografts established i n mi ce, respectively, were reported by one of the present i nvent ors and co-workers to exhi bit kill i ng of pediatric cancerous cell s. Swi ft et al . , Oncotargets Ther, 12:1293-1307 ( February 2019) .
I n addi ti on, i nt ral esi onal admi ni strati on of a hal ogenated xant hene compound into a tumor provi des the active cytotoxi c agent di rect I y to the tumor at its hi ghest concent rat i on. I n a presently cont empl at ed treatment technique discussed below, administration is often di stant from the target cancerous hematol ogi c cells, thereby possi bl y di mi ni shi ng the effectiveness of the canceroci dal hal ogenated xanthene compound medication ( agent) .
I n a phase I I cl i ni cal t ri al for pat i ents with refractory met ast at i c mel anoma, i nt r al esi onal

- 8 -i nj ect i on of PV- 10 aqueous RB di sodi um sol ut i on induced tumor regression with an overall response rate of 51% [Thompson et al . , Ann Surg Oncol 22( 7) : 2135-2142 ( 2015) ]. I nt ral esi onal PV- 10 aqueous RB di sodi um sol ut i on al so demonstrated ef f i cacy i n combi nat i on with radi ot her apy i n a phase I I cl i ni cal trial for pat i ents with i n- t ransi t or met ast at i c mel anoma, with an overall response rate of 86.6% [Foote et al . , J Surg Oncol 115( 7) : 891-897 ( 2017) ].
I n addition to i nduci ng di rect cancer cell death, PV- 10 aqueous RB di sodi urn sol uti on i nt r al esi onal admi ni strati on has al so been shown to induce a tumor-specific immune response in both mouse studies [ Qi n et al . , Cel I Death Di s 8:e2584 ( 2017) ;
Toomey et al . , PLoS ONE 8(7):e68561 ( 2013); and Liu et al . , Oncotar get 7( 25): 37893-37905 ( 2016) ] and i n human cl i ni cal trials [ Li ppey et al . , J Surg Oncol 114( 3) : 380-384 ( 2016); Ross, J Surg Oncol 109(4):314-319 ( 2104); Li u et al . , PLoS ONE 13( 4): e0196033 ( 2018);
and Basel et al . , Cancer Lett 412: 256-263 ( 2018) ].
I n muri ne model s of mel anoma, i nt r al esi onal treatment with PV- 108 aqueous RB di sodi um sol ut i on i nduced necrosis of mel anoma cell s and a I ocal i zed i ncr ease i n mononucl ear t umor- i nf iltr at i ng lymphocytes [ Li ppey et al . , J Surg Oncol 114(3) : 380-384 ( 2016) ] .
It has been suggested that PV- 10 aqueous RB
di sodi urn sol uti on i nduced i mmunogeni c cell death, rel easi ng tumor anti gens to nearby anti gen- present i ng cells (APCs) , and facilitated the activating of anti -tumor T and B cell s. I n a syngenei c muri ne col on cancer model, injection of cancer cells treated in

- 9 -vitro with PV- 10 aqueous RB di sodi um solution into mi ce with the same tumor resul ted i n slower tumor growth [ Qi n et al . , Cell Death Di s 8: e2584 ( 2017) ] .
Furthermore, i n syngenei c muri ne mel anoma model s, combi nation treatment with i nt r al esi onal PV- 10 aqueous RB di sodi urn sol ut i on and anti - PD-1 anti body del ayed tumor growth and enhanced T cel I act i vat i on [ Li u et al . , PLoS ONE 13( 4) : e0196033 ( 2108)].
Parental US Application No. 16/ 688, 319, filed on November 19, 2019, teaches that hematol ogi c cancer cell s such as I eukemi a cell s can be successful I y treated ( ki I I ed) by contact with an aqueous composi ti on contai ni ng a hal ogenated xanthene, a pharmaceutically acceptable salt, or a C1-C4 al kyl ester thereof.
Co-assi gned US Appl i cat i on Seri al No. 17/214, 590, f i I ed on March 26, 2021, teaches that solid cancerous tumors can be successfully treated by oral administration of a hal ogenated xanthene, its I act one, or a pharmaceut i call y acceptable sal t or ester thereof.
That oral I y admi ni stered medi cament coul d be i n sol i d or liquid form.
The di scl osure bel ow descri bes the contemplated invention and provides results of studies usi ng oral I y- admi ni stered hal ogenated xanthene compounds such as rose bengal i n the treatment of pediatric and adult leukemias.
BRIEF SUMMARY OF THE INVENTION
The present i nvent i on contempl ates a method of t r eat i ng a mammal i an subj ect havi ng I eukemi a.
The met hod comprises the steps of admi ni steri ng to such a

- 10 -mammal i an subj ect a t herapeut i cal I y ef f ect i ve amount of a hal ogenat ed xant hene ( HX) , a I act one, a pharmaceut i call y acceptable salt, or a C1-C4 al kyl or aromatic ester thereof ( col I ect i vel y referred to her ei n as an "HX compound") as a f i rst I eukemi a cytotoxi c agent dissolved or dispersed i n a pharmaceutically acceptable diluent solid or liquid medium. A
contempl at ed admi ni strati on is typically repeated.
A contemplated treatment met hod can al so be car r i ed out i n con] unct i on with admi ni strati on to that same mammal i an sub] ect of a second therapeutically effective amount of a second, differently-acting systemic leukemia cyt ot oxi c agent dissolved or dispersed i n a pharmaceutically acceptable medi urn.
The second systemic leukemia cyt otoxi c agent can be a small molecule, i oni zi ng radi at i on, or an i nt act anti body or paratope- contai ni ng anti body portion such as those protei naceous anti body mol ecul es that i nhi bit i nf I ammat ory chemoki ne activity or i mmune checkpoi nt anti bodi es. The f i rst and the second leukemia cytotoxi c agents can be admi ni stered together i n the same or different medi um, or i n the same or different medi urn at different ti mes. The second I eukemi a cytotoxi c agent can be admi ni stered i n a sol i d tabl et, capsul e, pi I I or t he I i ke, in a Ii qui d medi um, or as an intravenous i nj ect i on or i nf usi on.
In one aspect, use of a small-molecule I eukemi a cytotoxi c agent havi ng a mol ecul ar wei ght of about 200 to about 1000 Da i s contempl at ed. Compounds that synergi ze with a HX Compound such as doxorubi ci n, et oposi de and vi ncr i st i ne are preferred. I nt act

- 11 -anti bodies or paratope- contai ni ng anti body port i ons are a second group of leukemia cytotoxi c agents.
Preferred among these agents are those referred to as immune checkpoi nt i nhi bi tors.
[ See, for exampl e, Darvi n et al . , Exp Mol Nled, 50: 165 ( 2018) . ]
The present i nventi on al so contempl at es use of a therapeutically effective amount of an HX compound as a first leukemia cytotoxi c agent dissolved or dispersed i n a pharmaceutically acceptable aqueous medi urn for treatment of a mammal i an subj ect havi ng leukemia, wherein the halogenated xanthene compound ( HX
compound) i s mai ntai ned i n the mammal i an subj ect for a period of ti me suff i ci ent to i nduce death of leukemia cell s. In a further embodi ment, the f i rst I eukemi a cytotoxi c agent HX compound i s rose bengal , a pharmaceutically acceptable salt, I act one, or C1-C4 al kyl or aromatic ester thereof.
In a still further embodiment, the HX compound is rose bengal di sodi um salt.
Further, the typi cal I y treated leukemia cells are acute B- cel I or T- cel I I ymphobl asti c leukemia cell s, chronic I ymphocyti c I eukemi a cell s, or acute myel oi d I eukemi a cell s.
That any cancer, I et al one a sal i d cancerous tumor of the GI tract I i ke col orectal cancer, coul d be affected i n any way by an oral I y- admi ni stered HX
compound as di scl osed i n US Appl i cat i on Seri al No.
17/ 214, 590, filed on March 26, 2021, was quite unexpected because of I ow HX compound bi oavai I ability, f i rst- pass I asses of the drug, and al so because of the relatively short circulatory half-time ( about 30 mi nut es) previ ousl y reported for HX compounds such as

- 12 -rose bengal (RB) in other contexts. It was thus unexpected that oral admi ni strati on of rose bengal di sodi um, an illustrative HX compound, in a pharmaceuti call y acceptable sal t form di ssol ved i n an aqueous di I uent, coul d sl ow the progress of col orectal tumor devel opment i n ani mal s speci all y bred to devel op col orectal tumors i n the absence of any treatment.
It was even more unexpected that an orally-delivered contemplated HX compound could prevent formation of a col orectal cancerous tumor i n those specially bred ani mal s.
That an orally-administered effective amount of an HX compound coul d al so effectively kill I eukemi a cell s, as di scl osed herei n, was sti I I more unexpected for the reasons di scussed above, pl us the fact that leukemia cells are di stri but ed through out the body i n the blood stream as well as in the bone marrow. Thus, leukemia cells provide I ower concent rat i ons of more diffuse targets for the leukemia cytotoxi c HX compound to "f i nd" and be taken- up than are the cell s of a solid tumor that are rel at i vel y more concentrated and di rect I y fed by the tumor's arteries or are contacted by i nt ral esi onal admi ni strati on di rect I y into the tumor.
BRIEF DESCRIPTION OF THE FIGURES
I n the drawi ngs formi ng a part of thi s di scl osure:
Fi g. 1 is a graph showi ng survi val of CB17 SCI D mice from Charles River Laboratories International, I nc. , treated with oral I y- admi ni stered rose bengal di sodi um.
Exponent i all y- growi ng SEM cell s

- 13 -(2. 5x106 human ALL cells label I ed with GFP) were injected i nt ravenous! y i nto each ani mal and the establishment of tumors was monitored. After 4 weeks to permit the growth of the tumor, mice were randomized to three groups. Group 1 (n=9 control animals) received 100 [11_ of PBS given orally twi ce a week for two weeks. Group 2 ( n=8 treatment Cohort I ani mal s) recei ved 25 IAL of rose bengal di sodi urn present at 10 %
w/v i n 0.9 % NaCI aqueous sol uti on that was di I uted i n PBS to a f i nal vol ume of 100 IAL and given oral I y twi ce a week for 2 weeks. Group 3 ( n=8 treatment Cohort I I
animals) received 12.5 iit of the above 10 % w/v in 0.9 %
NaCI aqueous sol uti on that was di I uted i n PBS to a f i nal vol ume of 100 tiL and admi ni stered oral I y twi ce a week for 2 weeks. [vi dence of di sease progressi on was moni tored i n all ani mal s and survi val was f ol I owed up to 120 days f ol I owi ng the i ni ti at i on of treatment.
Data are presented as Kapl an-Mei er esti mates.
Exami ni ng the regi on between 50 and 100 days, the I i ne nearest to the X-axis represents data for the controls, the mi ddl e I i ne represents data for the Cohort I I
ani mal s, and the topmost I i ne represents data for the Cohort I ani mal s.
Fig. 2is a log-log plot of data from several different studies that plots the log of the rose bengal concent rat i on admi ni st ered ( mol an i ty) versus the I og of the duration of the HX compound in the subject up to the ti me of assessi ng solid tumor treatment, and i s al so present i n an earl i er form i n US Appl i cat i on Seri al No. 17/214, 590, filed on March 26, 2021.
"I nt r al esi onal Admi ni strati on" represents data present

- 14 -i n Thompson et al . , Mel anoma Res 18: 405- 411 ( 2008);
"Swi ft 2018, 2019" are from Swi ft et al . , J CI i n Oncol 36: Suppl ; abstr 10557 ( 2018) and Swift et al Oncotar gets Ther 12:1293- 1307 ( 2019); "Oral Apcivii n" are data from the study reported i n US Appl i cat i on Seri al No. 17/ 214, 590; and "Oral Leukemia" are new data presented i n the present appl i cat i on.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
I n one aspect, the present i nvent i on contempl at es an oral I y admi ni st ered pharmaceut i cal composi ti on for use i n treatment (kill i ng) of I eukemi a cell s present i n a mammal i an subj ect . A pr i nci pl e cytotoxi c agent in that oral pharmaceutical composition is a hal ogenated xanthene ( HX), the I act one thereof, a pharmaceutically acceptable salt thereof, or a C1-C4 al kyl or aromatic ester thereof, that are collectively ref erred to herei n as an "HX compound" that i s present in a I eukemi a- t reat i ng effective amount. An oral I
y admi ni stered pharmaceut i cal composi ti on can be i n solid or liquid form.
A contempl at ed hal ogenated xanthene mol ecul e i ncl udes rose bengal ( 4, 5,6, 7- t et rachl oro-2' , 4' , 5' , 7' -tet rai odof I uorescei n; RB) that is particularly preferred, eryt hrosi n B, phi oxi ne B, 4, 5, 6, 7-t et rabromo- 2' ,4' , 5' , 7' - t et r a- i odof I uorescei n, 2', 4, 5, 6, 7- pent achl oro-4' , 5' , 7' - t ri i odof I uorescei n, 4, 4' , 5, 6, 7- pent achl oro- 2' , 5' , 7' -tri i odof I uorescei n, 2' , 4, 5, 6, 7, 7'- hexachl oro- 4' , 5' - di i odof I uorescei n, 4, 4' , 5, 5' , 6, 7- hexachl oro- 2' , 7' - di i odof I uorescei n, 2' , 4, 5, 5' , 6, 7- hexachl oro- 4' , 7' - di i odof I uorescei n,

- 15 -4, 5, 6, 7- t et rachl or o- 2' , 4', 5' -trii odof I uorescei n, 4, 5, 6, 7- t et rachl or o- 2' , 4', 7' - t r i i odof I uorescei n, 4, 5, 6, 7- t et rabromo- 2', 4', 5'- tri i odof I uorescei n, and 4, 5,6, 7- t et rabromo- 2' ,4' , 7'- tri i odof I uorescei n.
The reader is directed to Berge, J. Pharm.
Sci . 1977 68( 1) : 1- 19 for lists of commonly used pharmaceutically acceptable acids and bases that form pharmaceuti cal I y acceptable salts with pharmaceuti cal compounds, such as the above halogenated xanthenes.
Ill ust rat i ve cat i ons i ncl ude al kal i metals such as sodi um, pot assi um, as well as ammoni um and al kal i ne earth salts such as magnesi um and cal ci um. The di sodi um sal t of rose bengal i s part i cul arl y preferred.
The I act one form of a contempl ated halogenated xanthene can be formed synthetically and is a preferred precursor of very pure rose bengal . I
n addi ti on, the carboxyl i c aci d form of a hal ogenated xanthene salt spontaneously forms the I actone form when in a strongly acidic aqueous environment such as that present i n a mammal i an stomach. When formed i n a mammal i an stomach or si mi I ar I y aci di c aqueous medi urn from the carboxyl i c acid or carboxyl ate salt form, the I act one not only forms, but al so appears to aggregate into clumps that do not readily dissolve in the duodenum and ad] acent small i nt est i nal regi on or i n an aqueous medi um havi ng a duodenal pH val ue.
A C1-C4 al kyl ester of one of the above halogenated xanthene compounds can al so be used, with the C2; i . e. , ethyl ester, bei ng preferred. In vitro studies using each of RB, ethyl -Red 3 (erythrosi ne ethyl ester; 2' , 4' , 5' , 7' - t et rai odo- f I uorescei n ethyl

- 16 -ester) , 4, 5, 6, 7- t et rabromo- 2' , 4' , 5' , 7' - tet rai odo-f I uorescei n, and ethyl - Phl oxi ne B ( 4, 5,6, 7- t et rachl or o-2' , 4' , 5' , 7' - t et rabromo- f I uor escei n ethyl ester) exhi bit Si mi I ar anti -tumor activities agai nst CCL- 142 renal adenocarci noma.
A contemplated aromatic ester is formed by a reaction between an HX molecule and an aromatic alcohol havi ng a 5- or 6- membered aromati c ri ng ( i ncl udi ng benzyl al cohol ), or a 5, 6- or 6, 6- f used aromat ic ri ng system that contai ns 0, 1 or 2 het ero ri ng atoms that are independently nitrogen, oxygen or sulfur. When an aromatic ester is used, it is preferably a benzyl , phenyl , or a 2-, 3-, or 4- pyri dyl ( pyri dyl ) ester, other aromat i c single and fused ring-containing esters are contemplated as discussed hereinafter. It is to be understood that although a benzyl ester is of ten consi dered to be an "aral kyl ester", for the purposes of t hi s i nventi on, a benzyl ester is deemed an aromatic ester.
I I I ust rat i ve examples of such aromatic alcohol ester port i ons are shown and named bel ow, where 0 is an oxygen atom and I i ne-0 indicates the ri ng-oxygen can be from any avail abl e carbon of the ri ng and the 0-li ne crossed by a wavy I i ne indicates that the depi cted al koxy group is a portion of another mol ecul e, the ester i f i ed HX molecule.
c\-535.
sV. ,s c-r\ ) N N
thienyl furyl oxazolyl thiazolyl

- 17 -csS"5 ¨ ¨0 naphthyl quinolyl quinoxalinyl >
benzofuranyl benzo[b]thienyl benzoxazinyl Rose bengal i s a preferred HX mol ecul e and its di sodi um sal t, rose bengal di sodi um, i s a most pref erred HX compound. A structural f ormul a of rose bengal di sodi um i s shown bel ow:
CI COONa Na0 0 0 Further detail s of the medi ci nal use of a pharmaceutical composition containing an above-noted HX
compound are descri bed i n U. S. Patents No. 5,998, 597, No. 6, 331, 286, No. 6, 493, 570, No. 7, 390, 688, No.
7, 648, 695, No. 8, 974, 363, No. 9, 107, 887, No. 9, 808, 524, No. 9, 839, 688, No. 10, 130, 658 and No. 10, 471, 144, whose di scl osures are i ncorporated by reference herei n i n thei r ent i reti es.

- 18 -Dosing - Fig. 2 Upon exposure of tumor cell s in a 0.9 %
sodi urn chl or i de- contai ni ng aqueous medi urn to an HX
compound, i rreversi bl e accumul at i on of the HX compound occurs i n tumor I ysosomes, causi ng i mmunogeni c tumor autol ysi s once a suff i ci ent concent rat i on i s achi eyed to destabilize I ysosomal i ntegri ty [Wachter et al . , SPI E 4620: 143-147 ( 2002) ] . Thi s suggests that t hi s i mmunogeni c mechani sm of cel I death can be el i cited over a range of exposure conditions based on a ( concent rat i on) = (time f unct i on) , where cyt ot oxi city is proport i onal to the product of these two parameters [ i . e. , cytot oxi city = f( [ HX] =t) , where "t" is time].
For exampl e, when RB i s admi ni stered in vi vo by i nt ral esi onal i nj ect i on to a range of sol i d tumors ( e. g. , melanoma, hepatocel I ul ar carci noma, breast carci noma) acute tumor cytot oxi city i s evi dent wi t hi n approximately 30 mi flutes for i nt rat umoral RB
concent rat i ons of approximately 25-50 mg/ g tumor tissue ( 25-50 mM) [ Thompson et al, Melanoma Res 18: 405-411 ( 2008) ].
Swift et al . [Oncotargets Ther 12:1293-1307 ( 2019) ] demonstrated cytotoxi city of treatment-ref ractory pediatric solid tumors (neurobl astoma and neuroepi t hel i oma) upon in vitro contact with RB for 96 hours at concent rat i ons of approximately 50-100 M.
Addi ti onal I y, Swi ft et al . , [J CI i n Oncol 36: Suppl ;
abst r 10557 ( 2018) ] , showed cyt ot oxi city in addi ti onal treatment- refractory pedi at r i c solid tumors ( Ewi ng sarcoma, osteosarcoma and rhabdomyosarcoma) under equi val ent exposure.

- 19 -Extended exposure to RB i n the context of conti nuous oral f eedi ng has been shown to prevent format i on of col on cancer ( prophyl act i c act i vi ty) and to arrest col on cancer (therapeutic activity) i n the muri ne ApcMi n col orect al tumor model as di scl osed i n parental US Appl i cat i on Seri al No. 17/214590, f i I ed on March 26, 2021. For t herapeuti c use, symptomatic mi ce recei vi ng RB ad Ii bi turn i n dri nki ng water at a concent rat i on of 1 mg/ mL had an approxi mate 38%
increase i n mean survi val relative to untreated mi ce ( 12. 3 O. 5 weeks vs 9. 8 O. 8 weeks) . Presumi ng a daily dr i nki ng water consumpti on rate of approxi mate! y 2 mL/ 10 g body wei ght, this corresponds to consumption of approximately 2 mg RB/l0 g ( 200 mg/ kg) .
Bi oavai I ability of RB di sodi um admi ni stered in aqueous solution via the oral route appears to be limited based on mass balance studies conducted by the inventors, and can be esti mated at O. 1- 1 percent, correspondi ng to a dai I y systemi c exposure of O. 2-2 mg/ kg. Presumi ng this amount is distributed through the bl oodst ream, and that bl ood vol ume comprises approxi mate! y 10 percent of body wei ght, t hi s equates to an esti mated concent rat i on of 2-20 M RB in the bl ood.
Thi s same approach was used to pl ot data presented i n Fi g. 1 of the present appl i cat i on, which shows survi val of CB17 SCI D mi ce with establ i shed xenografts of a pedi at ric B acute I ymphobl asti c leukemia (ALL) tumor cel I I i ne; therapeutic act i vi ty was observed for mi ce i n two treatment groups recei vi ng RB by gavage twice weekly for two consecutive weeks.

- 20 -Assuming 1% bi oavai lability of this oral RB, an i nt est i nal transit ti me of 6 hours per admi ni strati on, and a bl ood vol ume of approxi mate! y 10 percent of body weight, the two treatment groups correspond to an esti mated 125-250 pril RB i n the blood.
PI otti ng these data conf i rm that the hypothesized relationship ( i . e. , cytotoxi city =
f( [ HX] =t)) is supported by experimental results, as illustrated in Fig. 2.
More importantly, this functional rel at i onshi p permits predi ct i on of dose level and schedule appropriate to achi eve either an anti-tumor t her apeut i c outcome upon systemi c admi ni strati on.
For extended systemic treatment schedules equivalent to that i nvesti gated with the ApcMi n model, low mi cromol ar concent rat i ons ( i . e. , about 10 M) of ci rcul at i ng HX
compound are sufficient to achi eve I ysosomal accumulation and tumor cell destruction over a period of approxi mate! y 3 months, whereas mi cromol ar to submi cromol ar concent rat i ons ( i . e. , about 1 M) are suff i ci ent to achi eve tumor cel I destruction over a pen i od of approxi matel y 12 months.
Conversely, shorter durati on or i nterrupted repeat systemi c dosi ng at hi gher dose I evel , as used i n the present oral I eukemi a model , can al so achi eve tumor dest r uct i on.
For a speci fic i ndi cat i on, such as treatment of pedi at ri c pat i ents with I eukemi a, the rel at i onshi p of Fi g. 2 ill ust rates that standard approaches rout i nel y used by those of ski II in the art in pharmaceutical development can be applied to select an

- 21 -appropri ate dose I evel and schedul e that maxi mi zes therapeuti c outcome whi I e mi ni mi zi ng potent i al safety ri sk.
The ApcMi n data of Application Seri al No.
17/214590 and the oral I eukemi a treatment data of the present appl i cat i on show that a si mpl e f ormul at i on of the di sodi um salt of RB is suff i ci ent to deliver a therapeutically active level of RB; however, this may be I ess than ideally eff i ci ent as to bi oavai lability.
Determi ni ng a suitable f ormul at i on to achi eve eff i ci ent liberation and absorption of an orally delivered HX
compound i s thus a matter of standard pharmaceuti cal devel opment fami I i ar to those of ski II in the art, where the properties of the formulation can be varied to achi eve desi red bi oavai I ability by control of liberation ( di si ntegrat i on, di saggregati on and di ssol uti on) at an appropri ate poi nt wi thi n the GI
tract so as to maximize absorption of the dissolved HX
compound i nto the bloodstream.
Formulary optimization can be guided by standard pharmacoki net i c study of absorption such that dose I evel and formul at i on are ad] usted to achi eve the necessary systemi c exposure on the desi red dose schedule ( e. g. , about 100 1.011 in the bloodstream for short durati on exposure on the order of several days, about 1 to about 10 IP for i ntermedi ate durati on exposure on the order of several months, to about < 1 plvl or lower for long-term exposure on the order of a year or more).
The di basic salt forms of the HX compounds exi st i n sol uti on havi ng a pH greater than

- 22 -approxi mate! y 5, whereas at pH val ues < 5 the HX
compounds spontaneously convert to their I actone form.
Because the di basi c salt forms are hi ghl y sol ubl e i n aqueous medi a, whereas the 1 act one forms are i nsol ubl e in aqueous media, the former exhibit hi gher bi oavai 1 ability i n the GI tract compared to the latter.
Thus, opt i mi zi ng f ormul at i on to properly compensate for the pH val ue of the GI tract i s perhaps the most important parameter aff ecti ng bi oavai I ability. For exampl e, i n the stomach, where pH val ue < 4, di ssol ved HX compound is rapidly converted to the insoluble 1 actone form. Once i n the 1 act one form, the HX
compounds exhi bit hysteresi s and hi nderance to saponification back to the absorbable salt form, del ayi ng or i nhi bi ti ng downstream bi oavai lability.
However, the i ntral umi nal pH val ue rapi dl y i ncreases from hi ghl y aci di c in the stomach to around pH 6 i n the duodenum, and further i ncreases i n the small i nt est i ne from pH 6 to about pH 7.4 i n the termi nal ileum; pH drops to 5.7 i n the caecum before gradual I y i ncreasi ng to pH 6.7 i n the rectum.
[ pubmed. ncbi . nl m. ni h. govt 10421978/. 1 Thus, by appl yi ng standard means in the art of pharmaceutical formulation to achi eve i nt est i nal I i berati on, where the f avorabl e pH val ues facilitate HX compound I i berati on i n a di ssol vabl e, absorbabl e di basi c sal t form, bi oavai I abi I i ty is optimized.
For a 12-month treatment regi men, these data i ndi cate that a target concent rat i on of approxi mate! y 1 iiM ( 1 mg/ L) i s achi eyed i n the bl oodst ream. For a kg adul t human, where bl ood vol ume compri ses approximately 10% of body weight ( i . e. , about 7 L),

- 23 -this implies absorption of 7 mg HX compound/ day.
If bi oavai lability is limited to 1% of admi ni stered HX
compound, then 700 mg HX compound per os (P0) woul d be requi red daily to achi eve t hi s target bl ood I evel .
However, by opt i mi zi ng absorpti on to 50% of admi ni stered dose, the necessary PO dose i s reduced to approximately 15 mg daily. For a shorter treatment regi men ( i . e. , 3 months), these data i ndi cat e that a target blood concentration of approximately 10 M ( 10 mg/ L) is achi eyed.
Presumi ng 1% bi oavai lability, then 7 g HX compound PO i s requi red dai I y, whereas at 50%
bi oavai lability, the necessary dose i s reduced to approxi mate! y 150 mg dai I y.
One contempl at ed pharmaceutical composition compri ses a O. 1 % to about 20 % (w/v) aqueous medi um (as a I i qui d) of a f i rst leukemia cytotoxi c agent that is a halogenated xanthene compound ( HX compound). More preferably, that concent rat i on is about 0.2 to about 10 'Yo ( w/v), most preferably, the concent rat i on is about 0.2 to about 5 % ( w/v). Thus, for example, the above dose of 150 mg dai I y coul d readily be achi eyed by use of 3 mL of a 5 % ( w/v) aqueous solution.
A part i cul ar I y preferred halogenated xant hene salt is rose bengal ( 4, 5, 6, 7- t et rachl oro- 2' , 4' , 5', 7' -t et rai odof I uorescei n) di sodi um ( RB di sodi um) salt.
The pharmaceuti cal composi ti on i s admi ni stered oral I y to provi de a therapeutically effective amount of a f i rst leukemia cytotoxi c agent to a mammal such as a human havi ng leukemia, or more specifically, acute I ymphobl asti c I eukemi a (ALL) as T- ALL or B- ALL, chronic I ymphocyti c leukemia (CLL), or acute myeloid leukemia (AML).

- 24 -The mammal i an subj ect i s typically treated mul ti pl e ti mes. The fact and relative amount of I eukemi a cell kill i ng can be determi ned by usual means for assayi ng the status of a gi ven I eukemi a mammal i an subjects. Both the duration of maintenance and the choice to conduct further admi ni strati ons can depend upon the speci es of mammal , i ndi vi dual mammal i an subjects, the severity of disease, type of disease, age and health of the subject, and the observed effect on the burden of leukemic cells caused by the treatment.
These factors are commonly dealt with by physicians ski I I ed i n the art of t reat i ng leukemia.
I n addi t i on, whereas it is typi cal I y desi red to rid the body of detect abl e I eukemi c cell s, that cannot al ways be done. Sometimes it is sufficient to kill enough I eukemi c cel Is to control the di sease i n stasis, or to reduce the leukemic load of cells so that other therapies can be carried out.
The data provi ded herei naf ter ill ust rate that the I Cso val ue for use of RB agai nst several I eukemi a cell lines in vitro is about 50 to about 100 pLM for exposures of one to several days. Given that the molecular weight of RB di sodi um is 1018 g/mol e, the above I Cso value calculates to about 50 to about 100 mg of RB/ I i ter. It is preferred to achi eve that concent rat i on for contact i ng I eukemi c cells dun i ng an in vivo treatment.
The cl assi c i nt ravenous ( I V) di agnost i c assay for liver function usi ng RB was conducted gi vi ng 100 mg RB as a single IV dose.
In clinical studies of PV- 10 aqueous RB di sodi um sol ut i on, RB has been t ol erated at

- 25 -1500 mg deli vered IV. The standard adul t bl ood vol ume is approximately 5 L.
Thus, to achieve 100 mg/ Lint he blood, an adult patient would need to receive approxi mate! y 500 mg of RB IV to achi eve the I C50 val ue in the bloodstream.
Due to the rapid clearance of RB
from circulation ( t112 is about 30 minutes), an IV
admi ni strati on can requi re cont i nuous i nf usi on to mai ntai n peak I evel s of RB i n ci rcul at i on ( i . e. , for up to several hours or more) .
Administration at the I C50 val ue level would not be toxi c to all ci rcul at i ng hematol ogi c, non-tumorous I eukemi c cell s; i . e. , onl y approxi matel y hal f of cells would be affected at the I C50 val ue.
It can therefore be preferred to admi ni ster RB at a multi pl e of the I C50 value, up to approximately 1500 mg ( i . e. , 300 tiM).
Alternatively, it can be suf f i ci ent to kill only a f ract i on of the I eukemi c cells to initiate a f uncti onal i mmune response agai nst remai ni ng I eukemi c burden. The latter case can be preferable to avoid toxi c react i on ( i . e. , so- cal led "tumor I ysi s syndrome") due to presence of an abundance of rapidly killed leukemia cells. I n t hi s Si tuat i on, the I eukemi a cell debri s caused by the cytotoxi city to I eukemi a cell s of a hal ogenated xant hene rel eases i ntracel I ul ar contents such as pot assi um, causi ng non- speci f i c cell death.
This process may al so activate the immune system specifically agai nst the mal i gnant cells.
The similarly useful hal ogenated xanthene compounds previ ousl y- I i sted and thei r pharmaceuti cal I y acceptable salts can have molecular weights that differ

- 26 -from each other by about a factor of three ( See, Table 3, US Patent No. 7, 390, 688 at col umns 15- 16) . It is preferred that an exact amount of other than RB
halogenated xanthene to be used is calculated based on publ i shed mol ecul ar wei ghts for each such compound and that of RB or RB di sodi um.
A mammal i an subj ect havi ng I eukemi a i n need of treatment (a mammal i an subj ect) and to which a pharmaceutical composition containing a halogenated xanthene compound can be admi ni stered can be a pri mate such as a human, an ape such as a chi mpanzee or gori I I a, a monkey such as a cynomol gus monkey or a macaque, a I aboratory ani mal such as a rat, mouse or rabbit, a companion ani mal such as a dog, cat, horse, or a food ani mal such as a cow or steer, sheep, I amb, pig, goat, llama or the like.
I n one aspect of the i nventi on, a contempl ated HX compound for oral admi ni strati on i s typi call y used di ssol ved or di spersed i n a steri I e aqueous pharmaceutical composition.
Sterile tap water or sterile water from another source can be used.
Characteristics of a Contemplated Liquid Pharmaceutical Composi ti on An HX compound i s typi call y present i n a contempl ated aqueous pharmaceut i cal composition at about 0.1 to about 20 % ( w/v). More preferably, that concent rat i on i s about O. 2 to about 10 % (w/v), most preferabl y, the concent rat i on i s about 0.2 to about 5 %
(w/v). Thus, for example, the above dose of 150 mg dai I y coul d readi I y be achi eyed by use of 3 mL of a 5 %
(w/ v) aqueous sol ut i on.

- 27 -The bi oavai I ability of van i ous HX compounds such as di sodi urn rose bengal has not been well characterized. Studies commissioned by one of the assi gnees concl uded that bi oavai I ability of di sodi urn rose bengal is less than (<) 1% based on radi ol abel studies where 14C- RB in aqueous solution was given oral I y to mi ce.
I n the stomach, with a pH val ue < 4, an HX compound is Ii kel y to be i n the I actone form.
Conversi on to I act one i n the stomach does not destroy the HX compound, but such conversi on i nto the I act one form can present a ki net i c and/or thermodynamic barrier to reconversi on to the sol ubl e sal t form necessary for absorpti on i n the i nt est i nes.
The study di scussed i n U.S. Patent Application Seri al No. 17/214590 using Apcmi n mi ce showed that those mi ce consumed 4 mg/ mL ad Ii bi turn i n dri nki ng water arrested onset of disease. Those Apcmi n mi ce are understood to have thereby consumed approxi mate! y 8 mg/ 10 g/ day = 800 mg/ kg/ day. That amount i s consi stent with toxi col ogy data showi ng such a dose i s t ol erat ed.
Thus, I to et al . , J Nat! Cancer 1, 77: 277- 281 ( 1986) studying rose bengal as a food col or i ng ( Food Red No. 105) found that rose bengal fed ad libitum continuously for 2 years at a dose of 970 mg/ kg/ day to C57BL6N mi ce was well tolerated. The previ ousl y used i nt ravenous (I V) I i ver di agnosti c deli vered 112 mg of rose bengal as a bol us; for a standard 60 kg adult human, which equates to 1.9 mg/ kg;
this has not reported to yield morbidity.
It is preferred that a liquid pharmaceuti cal composi ti on for oral admi ni strati on have an osmol al i ty

- 28 -less than that of blood plasma.
Normal (well) human reference range of osmol al i ty i n pl asma i s about 275-299 mill i - osmol es per ki I ogr am (mOsm/ kg).
More preferably, that composition is free of toni city agents ( or toni city-adjusting agents) such as sugars I i ke manni tol and dextrose, C3-C6 pol yhydroxy compounds such as propyl ene gl ycol , gl ycerol and sorbi tol , i sotoni c salts such as sodi um or potassi um chl on de, and/or buff eri ng agents other than those such as citric acid, mal i c acid, acetic acid and other food aci ds and thei r salts that can be provi ded for fl avor and mild buffering (less than 5 mmol of buffering agent) . The stomach and I ower GI tract are well adapted to provi de the proper toni ci ty to materi al s fl owi ng through such that further salts and/or buffers are not needed. One or more pharmaceuti cal I y acceptable taste- maski ng agents or fl avorants as are well - known can be present at up to about 5% by wei ght to enhance the potability of the composi ti on.
It is preferred that the pH val ue of a pharmaceuti call y acceptable aqueous di I uent be about 5 to about 9, to yield maxi mum sol ubi I i ty of the HX
compound i n an aqueous vehi cl e and assure compati bi I i ty with biological ti ssue.
A particularly preferred pH
val ue is about 5 to about 8, and more preferably between about 6 to about 7.5. At these pH val ues, the hal ogenated xanthenes typi call y remai n i n di basi c form, rather than the I actone that forms at low pH val ues.
An HX compound such as rose bengal i s di basi c, havi ng pKa val ues of 2.52 and 1.81.
pKa val ue determi nati ons for several contempl ated hal ogenated

- 29 -xant henes can be found i n Batsi tel a et al . , Spectrochi m Acta Part A 79( 5) : 889-897 (Sept. 2011) .
I n the present i nvent i on, the specific amount of halogenated xanthene compound in a pharmaceutical composi ti on i s not bel i eyed to be as i mport ant as was the case where the composition was injected i nt ral esi onal ly to a tumor because the object here is to ul ti matel y provide a cytotoxi c concent rat i on of hal ogenated xanthene compound to the envi ronment of the I eukemi c cell s and i n whi ch those I eukemi c cell s can be contacted with the halogenated xanthene compound. The data provi ded herei nafter i ndi cat e that an I CH
concent rat i on of di sodi um rose bengal is about 50 to about 100 M for in vitro cultured leukemia cells.
The above-noted results using in vitro cul t ured I eukemi a cell s surpri si ngl y provided data si mi I ar to those obtai ned i n an in vitro cyt otoxi city study of cul t ured SK- N- AS, SK- N- BE( 2) , I MR5, LAN1, SHEP, and SK- N- SH neurobl ast oma cells, SK- N- MC
neuroepi t hel i oma cell s, and normal pr i mary, Bj , and WI 38 f i brobl asts reported by Swift et al . , OncoTargets and Therapy 12: 1-15 ( 2019). Those authors reported half maxi mal inhibitory concentration ( I C50) val ues for PV- 100 aqueous RB di sodi um sol ut i on-treated cells at 96 hours post treatment of 65-85 M for the neurobl astoma I i nes assayed and 49 M for the neuroepi thel i oma I i ne SK- N- MC. Those authors al so examined toxicity toward human epithelial cells from three tissue sources and reported I C50 values of 93-143 M.

- 30 -I n ci i ni cal studi es of PV- 10 aqueous RB
di sodi um sol uti on, RB has been tol erated at 1500 mg delivered I L. Due to the rapi d ci earance of RB from ci rcul at i on (t112 about 30 mi nutes) an IV
admi ni strati on can requi re cont i nuous i nf usi on to mai ntai n peak I evel s of RB i n ci rcul at i on ( i . e. , for up to several hours or more) dun i ng a si ngl e admi ni strati on.
Characteristics of a Contemplated Solid Pharmaceutical Composi ti on It is further contempl ated that the HX
compound such as RB or di sodi um RB, or a HX compound I act one such as RB I act one be admi ni stered i n a sol i d pharmaceuti cal composi ti on for oral admi ni strati on that is enteri cal I y- coated to pass through the stomach and rel ease the HX compound i n the i ntesti nes. The HX
compound i s typi call y di ssol ved i n or di spersed i n or on a sol i d di I uent medi um.
There are several factors at play in the di ssol uti on of an oral I y admi ni stered sol i d pharmaceuti cal product i n a mammal i an body. Among those factors are resi dence ti me of the medi cament at different locations along the GI tract, particle size, sol ubi I i ty of the i ndi vi dual components of the medi cament i n the bodily fl ui ds I i kel y to be encountered from mouth to anus, the order i n which van i ous coati ng layers, when present, are applied to the medi cament, as well as the pH val ue at whi ch a part i cul ar coati ng layer is sol ubl e.

- 31 -For example, the highly aci di c gastri c envi ronment ( pH 1.5-2 in the fasted state; pH 3-6 in the fed state) ri ses rapi dl y to about pH 6 i n the duodenum and i ncreases al ong the smal I i ntesti ne to pH
7.4 at the termi nal i I eum. The pH value in the cecum drops j ust bel ow pH 6 and agai n ri ses i n the col on reachi ng pH 6. 7 at the rectum [ Hua, Front Pharmacol 11: Art i cl e 524 (April 2020)].
Observation of sol uti ons of di sodi urn RB mixed i nto a water sol uti on havi ng the pH val ue of the human stomach reveal ed rapi d cl oudi ng of the admi xture and cl umpi ng of the previously sol ubl e di sodi um RB, presumably i nto the I act one form.
Gastric transit can range from 0 to 2 hours i n the fasted state and can be prolonged up to 6 hours in the fed state. I n general, the transit time i n the small i nt est i ne is consi dered relatively constant at around 3 to 4 hours, but can range from 2 to 6 hours i n heal thy i ndi vi dual s. Col oni c transit times can be highly variable, with ranges from 6 to 70 hours reported [ Hua, Front Pharmacol 11: Art i cl e 524 (April 2020) ] .
Drugs must pass or permeate through the epi t hel i al cel I s t hat I i ne t he i nner wal I s of t he GI
tract i n order to be absorbed i nto the ci rcul atory system.
A cell ul ar barni er that can prevent epi t hel i al cell absorption of a given drug is the cell membrane.
Cell membranes are essentially lipid bi layers that form a semi per meabl e membrane.
Pure I i pi d bi I ayers are general I y permeabl e onl y to small , uncharged sol ut es. Hence, whether or not a mol ecul e is i oni zed will affect its absorpti on, because i oni c mol ecul es are charged.
Sol ubi I i ty favors

- 32 -charged speci es, and permeabi I i ty favors neutral speci es.
Typi call y, i ons cannot passively diffuse through the gastroi ntesti nal tract because the epi thel i al cell membrane is made up of a phosphol i pi d bi I ayer.
The bi I ayer i s made up of two layers of phosphol i pi ds i n which the charged hydrophi I i c heads face outwards and the non-charged hydrophobi c fatty acid chai ns are i n the mi ddl e of the layer. The uncharged fatty aci d chai ns repel i oni zed, charged molecules.
This means that the ionized molecules cannot easi I y pass through the i ntesti nal membrane and be absorbed.
Chemi cal modification by est er i f i cat i on can be used to control sol ubi I i ty. For example, C2-C4 al kyl and aromati c ester forms of an HX compound typi call y have decreased sol ubi I i ty i n aqueous I i qui ds, and because of thei r neutral i oni c charge, are typi call y better taken- up by i ntesti nal epi thel i al cells than thei r carboxyl ate forms. Later, esterases i n the GI tract wall and bl ood hydrol yze these esters to release the parent drug.
Al so, coati ng f i I ms on a tab! et or a pel I et can act as a barn i er to reduce the rate of di ssol uti on and/or di si ntegrat i on of the composi ti on in aqueous media, generally, and particularly within the stomach.
A coati ng can al so be used to modify where di ssol uti on takes pl ace. For exampl e, enteri c coati ngs can be appl i ed to a drug- contai ni ng medi cament, so that t he coati ng and the drug only di ssol ve i n the basic envi ronment of the i ntesti nes. One approach useful for

- 33 -predictable rel ease of a drug from a medicament in the i ntesti nal portion of the GI tract and/or at a part i cul ar location i n the GI tract relies upon pH-specific coati ngs and mat ri ces that dissolve or di si ntegrate at presel ected GI tract pH val ues such as those noted previously.
The tabl e bel ow shows some exampl es of pH-dependent pol ymer coati ngs that have been used for the purpose of targeting rel ease (local treatment) either al one or i n combi nati on, i ncl udi ng some met hacryl i c resins (commercially available from Evoni k Industries, AG, Essen, Germany as Eudragi tO), and hydroxypropyl methyl cel I ul ose ( HPMC; available from DuPont, Wilmington, DE as Met hocel TM; and Ashl and, I nc. , as Benecel TN, W I I mi ngt on, DE) der i vat i yes.
I n addi ti on to tri ggeri ng rel ease at a speci f i c pH value range, the enteri c coati ng can protect the incorporated active agent agai nst the harsh GI tract envi ronment ( e. g. , gastri c j ui ce, bi I e aci d, and mi crobi al degradati on) and can create an extended and del ayed drug rel ease prof i I e to enhance therapeutic eff i ci ency.
The "publ i shed pH rel ease" val ue for each pol ymer is from the manufacturer. The "publ i shed pH
rel ease" val ues are not absol ute for all composi ti ons or envi ronments, and pH val ues for di ssol uti on or di si ntegrati on stated herei n are based on those publ i shed val ues.

- 34 -pH- Dependent Pol ymer Coat i ngs*
Pol ymer Publ i shed pH Rel ease Eudragi t 0 S-100 7.0 Eudragi t 0 FS-30D 7.0 Eudragi t 0 L-100 6.0 Cell ul ose acetate pht hal ate 6. 0 Cell ul ose acetate tri mel I i tat e 5. 5 Eudragi t L- 30D- 55 5. 5 Eudragi t 0 L- 100- 55 5.5 Hydroxypr opyl methyl cell ul ose pht hal ate 55 5. 5 Hydroxypr opyl methyl cell ul ose pht hal ate 50 5. 0 Pol yvi I yl acetate pht hal ate 5. 0 *[ Hua, Front Pharmacol 11: Arti cl e 524 (April 2020) 1 For colonic release, colon-targeted drug delivery systems have been actively pursued because convent i onal non-targeted therapy can have undesi rabl e side-effects and low efficacy due to the systemic absorption of drug before reaching the target site.
Li u et al . , Eur. J. Pharm. Bi opharm. 74: 311-315 ( 2010) , adopted dual coati ng approach by usi ng the al kal i ne aqueous sol uti on of Eudragi t S with bufferi ng agents for i nner I ayer and the organi c sol uti on of Eudragi te S
for outer I ayer, accel erati ng the drug di ssol uti on at pH val ues greater than 7. Subsequently, Varum et al Eur. J. Pharm. Bi opharm. 84: 573-577( 2013) , evaluated in vivo performance of this dual coated system in humans, demonst rat i ng more consi stent di si ntegrati on of dual coated tabl ets mai nl y in the lower intestinal tract.

- 35 -Hashem et al . , Br. J. Pharm. Res. 3: 420-434 ( 2013), developed mi cr ospher es combining time-and pH-dependent systems for col oni c deli very of predni sol one.
By usi ng a combi nation of Eudragi t S and ethyl cell ul ose, they achi eyed greater col oni c drug delivery, whi I e prevent i ng premature drug rel ease i n the upper i nt est i ne.
Eudracol i s another exampl e of a multi - unit technol ogy provi di ng targeted drug delivery to the colon, with delayed and uniform drug release. This system i s based on coati ng the pellet with Eudragi t RL/ RS and Eudragi t FS 30D, providing colon-specific drug rel ease in a pH-and time-dependent manner [ Patel , Expert Opi n. Drug Del i v. 8:1247-1258 ( 2011) ] .
One composi ti on that targets the small i nt est i ne comprises a di I uent medi um of sugar/sucrose beads coated with part i cul ate rose bengal ( RB) that i s coated with one or a pl ural i ty of layers of a ( met h) acryl ate copol ymer t hat i s composed of about 60 to about 95% by wei ght free radical polymerized C1-C4-al kyl esters of acryl i c or met hacryl i c aci d and about 5 to about 40% by wei ght ( met h)acryl ate monomers with an aci di c group i n the al kyl radi cal .
Parti cul arl y suitable (meth)acryl h) acryl ate copol ymers i ncl ude about 10 to about 30% by wei ght methyl met hacryl ate, about 50 to about 70% by wei ght methyl acryl ate and about 5 to about 15% by wei ght met hacryl i c acid ( Eudragi t FS type). Si mi I ar I y suitable, are ( met h)acryl ate copolymers of about 20 to about 40% by wei ght met hacryl i c acid and about 80 to about 60% by wei ght methyl met hacryl ate ( Eudragi t S

- 36 -type) .
The word "( met h) acryl ate" is used herein to mean that either or both of acryl ate and met hacryl ate monomers can be used.
These coati ng polymers permit little if any HX compound release prior to the particles leaving the stomach. The pH value of the fluid within the duodenum typically is about 6 and rises to about 7.4 toward the i I eum.
A usual tablet or lozenge can be prepared by admi xt ure of I act ose ( 20%) and act i ve i ngredi ent ( 80%;
HX compound) mixed in a high-speed mixer ( DI OSNA type P10, Osnabruck, Germany) . An aqueous sol uti on contai ni ng the exci pi ent pol yvi nyl pyrrol i done ( PVP) such as povi done ( Si gma- Al dr i ch International GmbH, Buchs, CH) i s added i n smal I amounts unti I a homogeneous composition is obtained. The moist powder mixture is screened. Tablets are subsequently made therefrom as is well-known, and dried.
The resul ti ng tabl et s or I ozenges are thereafter preferably coated with a protective pol ymer film, of ten usi ng fl ui di zed bed equi pment . Fi I m-f ormi ng pol ymers are normal I y mixed with pl asti ci zers and release agents by well-known processes. The film formers can in this case be in the form of a solution or suspensi on. The exci pi ents for the film formation can I i kewi se be di ssol ved or suspended.
Organi c or aqueous sol vents or di spersants can be used.
Stabilizers can be used i n addi ti on to stabilize the dispersion (for example: Tween 80 or other suitable emul si f i ers or stabilizers).

- 37 -Exampl es of rel ease agents are gl ycerol monostearate or other suitable fatty acid derivatives, si I i ci c aci d der i vat i yes or t al c. Exampl es of pl asti ci zers i ncl ude propyl ene gl ycol , pht hal at es, pol yet hyl ene gl ycol s, sebacates or ci t rates, and other substances mentioned above and in the literature.
Another preferred type of medi cament i s a water- sol ubl e capsul e or bl i ster that contai ns a pl ural i ty of part i cl es of an HX compound such as rose bengal di sodi um or rose bengal I act one that are covered with one or more I ayers of polymeric resi n that rel ease the HX compound quickly upon dissolution or di si ntegrati on of the capsul e i n water or body fl ui d.
Capsul es are typi call y made of gel at i n and are often ref erred to as gel caps.
Gel at in is an ani mal product.
Vegetari an capsul es are often made of hydroxypropyl methyl cel I ul ose ( HPMC).
I n some embodi ments, the HX compound is di rect I y layered with one or more coats of the pol ymer to form particles that are general I y spheri cal i n shape.
Such particles are often ref erred to as beads.
I n a preferred aspect, part i cl es ( beads) are si zed so as that about 90 percent by wei ght pass through a 20 mesh si eve ( openi ng = 850 m) screen and about 90 percent by wei ght are retai ned on an 80 mesh si eve (openi ng = 180 r11) screen.
Exemplary pH value-sensitive coating pol ymeri c resi ns are di scussed above. The pH val ue-sensi ti vi ty of coati ng pol ymeri c resi ns is to be understood i n terms of physi ol ogi call y present pH

- 38 -val ues al ong the GI tract such as those di scussed above.
I n other embodi ments, small pellets such as sugar/starch seeds, non- parei Is or pri I I s, which are small , general I y spherically-shaped cores, are coated with one or a plurality of layers of the HX compound and one or more layers of polymeric coating.
Illustrative sugar/starch cores are sugar spheres NF
that pass through an about 40 mesh si eve (425 mm openi ng) screen to an about 50 mesh si eve ( 300 mm openi ng) screen, that contai n not less than 62.5 percent and not more than 91.5 percent sucrose, calculated on the dry basis, the remainder consisting primarily of starch. ( USP NF 1995 2313).
I n an ill ust rat i ve example, a 100 kilogram ( kg) quantity of di sodi um rose bengal , a 7.1 kg quantity of cross- I i nked car boxymethyl cell ul ose (preferably croscarmel I ose sodi um NF), and an 11.9 kg quantity of starch NF, are each di vi ded i n half, and the three constituents are blended together to form two i dent i cal batches. Each of the batches is mill ed through an 80 mesh screen usi ng a mill such as a Fi tzpat ri ck Mill . The two mill ed batches are then bl ended to form a mixture, whi ch i s tested for composi ti on i n accordance with accepted quality assurance testing methods that are well-known by those skilled in the art.
The di sodi um rose bengal mi xture i s subsequently di vi ded i nto three equal parts, with a f i rst part remai ni ng whol e, and second and t hi rd parts each divided into lots of 50 percent, 30 percent and 20 percent. A 25.6 kg quantity of 40-50 mesh sugar/starch

- 39 -seeds (e. g. , sugar spheres NF) is placed in a stai nl ess steel coati ng pan. An 80 liter ( L) quantity of 5 percent povi done/ i so- propanol ( I PA) solution is prepared for sprayi ng onto the part i cl es.
The coati ng pan is started with the sugar spheres, onto whi ch i s sprayed an appl i cat i on (approximately 0.173 kg per appl i cat i on) of the povi done- al cohol solution, and onto which is sifted an application (approximately 0.32 kg) of the di sodi um rose bengal mixture from the first part (that part that remai ned whole). Si fti ng i s done usi ng a standard sifter. The sprayi ng and si fti ng steps are conti nued until the f i rst part of the mixture has been appl i ed to the sugar spheres to form a batch of partially coated spheres.
The partially coated spheres are then divided i nto two equal lots, each I ot bei ng pl aced i n a coati ng pan. Separately for each of the two I ots, sprayi ng of the povi done/ I PA solution and sifting of the di sodi um rose bengal mixture as di vi ded i nto the 50 percent I ots conti nues until the 50 percent lots have been applied to the spheres. Fol I owi ng appl i cat i on of the 50 percent lots, the spheres can be screened usi ng a 25 mesh screen if necessary.
The sprayi ng of the povi done/ I PA sol uti on and si fti ng of the di sodi um rose bengal mixture as divided into the 30 percent lots commences and conti nues unti I
the 30 percent I ots have been appl i ed to the spheres.
The coated spheres can be rescreened usi ng a 25 mesh screen.
Spraying of the povi done/I PA solution and si fti ng of the di sodi um rose bengal mixture conti nues

- 40 -usi ng the mixture as di vi ded i nto the 20 percent lots until the 20 percent lots have been applied to the spheres. At this poi nt i n the process, the enti re quantity of the di sodi urn rose bengal mixture has been appl i ed to the spheres, and about 50 kg of the 5 percent povi done/1 PA sol uti on has been applied to the spheres.
A 7.5 percent povi done/I PA solution is prepared and applied to the spheres as a sealant. The sealed spheres are tumble dried for about one hour, wei ghed, and pl aced i n an oven at about 122 oF ( 50 0C) for 24 hours. After dryi ng, the spheres are screened through a 20 mesh screen and a 38 mesh screen to form the immediate (quick or fast as compared to del ayed) rel ease particles.
The above-discussed HX compound-containing spheres or thei r capsule (or blister) can al so be coated with a pH value-sensitive enteri c coati ng pol ymer as di scussed previ ousl y so that once rel eased in the GI tract, the spheres do not provide thei r active i ngredi ent, HX compound, to thei r surroundi ngs unl ess the pH val ue i s at least that of a desi red GI
tract location.
Another way to control the 1 ocat i on of HX
compound rel ease is to further coat the spheres ( HX-coat ed part i Cl es) discussed above, with a di ssol uti on-control I i ng coat of pol ymeri c resi n applied to the surface of the spheres such that the rel ease of the HX
compound from the spheres i s controlled and rel eased over a 6-10 hour pen i od. The materials used for this purpose can be, but are not I i mi ted to, ethyl cell ul ose,

- 41 -hydroxypropyl methyl-cell ul ose, hydroxypropyl cell ul ose, methyl cell ul ose, hydroxyet hyl cell ul ose, ni t rocel I ul ose, car boxymet hyl - cel I ul ose, as well as copolymers of et hacryl i c acid and met hacryl i c acid ( Eudragi tg), or any other acrylic acid derivative (Carbopol g, etc.) can be used.
I n addi ti on, an enteri c coating material can al so be empl oyed, either si ngul ar I y, or i n combi nat i on to the above non- pH- sensi t i ve coatings. These mat er i al s i ncl ude, but are not I i mi t ed to, hydroxypropyl methyl cell ul ose pht hal ate and the pht hal ate esters of all the cel I ul ose ethers. I n addi ti on, pht hal ate esters of the acryl i c acid der i vat i yes ( Eudragi t ), or cel I ul ose acetate pht hal ate.
These coati ng mat er i al s can be empl oyed i n coati ng the surf aces i n an amount of about 1.0 percent ( w/ w) to about 25% ( w/w) . Preferably, these coati ng materials are present at about 8.0 to about 12.0 percent ( w/w) .
Exci pi ents Exci pi ents customary i n pharmacy can be empl oyed i n a manner known per se i n the product i on of the HX compound- contai ni ng medi cament . These exci pi ents can be present i n the core or i n the coati ng agent.
Pol ymers Pol ymer i c mat er i al s used as adhesi yes i n hel pi ng to adhere an HX compound to a sugar pri I I or sphere i s deemed to be an exci pi ent where coati ng layers of an HX compound are empl oyed.
Ill ust rat i ve of

- 42 -such poi ymers are poi yvi nyl pyrrol i done and poi yvi nyl alcohol as are other water-sol ubl e, pharmaceuti cal 1 y acceptable fi I m- f ormi ng pol ymers such as hydroxypropyl cel 1 ul ose.
Dryers ( non- sti ck agents) Dryers have the f ol I owi ng properti es: they have 1 arge specific surface areas, are chemically inert, are free-fl owi ng and compri se f i ne part i cl es.
Because of these properti es, they reduce the tack of polymers containing polar comonomers as functional groups. Examples of dryers are: al umi na, magnesi um oxi de, kaol i n, t al c, fumed si I i ca, bar i um sul phate and cell ul ose.
Di si ntegrants Di si ntegrants are added to oral sol i d dosage forms to ai d in thei r di saggregati on. Di si ntegrant are formul ated to cause a rapi d break- up of sol i ds dosage forms on contact i ng moi st ure. Di si ntegrati on is typically viewed as the first step in the dissolution process. Illustrative di si ntegrants include sodi um croscarmel I ose, an i nternal 1 y cross-1 i nked sodi um car boxymet hyl cell ul ose, cross- I i nked pol yvi nyl pyrrol i done ( crospovi done) and sodi urn starch gl ycol ate.
Release Agents Exampl es of rel ease agents are: esters of fatty acids or fatty ami des, al i phat i c, 1 ong- chai n carboxyl i c aci ds, fatty al cohol s and thei r esters, montan waxes or paraff i n waxes and metal soaps;
particular mention should be made of glycerol monostearate, stearyl alcohol, glycerol beheni c acid ester, cetyl alcohol, pal mi tic acid, carnauba wax, beeswax, and the I i ke. The usual proporti onate amounts

- 43 -are i n the range from 0.05 percent by wei ght to 5, preferably 0.1 to 3 percent by wei ght based on the copol ymer.
Other exci pi ents customary in pharmacy Menti on shoul d be made here of, for exampl e, stabi I i zers, col orants, anti oxi dants, wetti ng agents, pi gments, gl oss agents. They are typi call y used as processi ng ai ds and are i ntended to ensure a rel i abl e and reproducible production process and good long-term storage stability. Further exci pi ents customary i n pharmacy may be present i n amounts from 0.001% by wei ght to 10% by weight, preferably 0.1 to 10% by wei ght, based on t he pol ymer coati ng.
PI asti ci zers Substances suitable as plasticizers ordi nari I y have a mol ecul ar wei ght between 100 and 20, 000 and compri se one or more hydrophi I i c groups i n the molecule, e.g. hydroxyl, ester or amino groups.
Ci t rates, pht hal ates, sebacates, castor oil are sui table. Exampl es of further sui tabl e pl asti ci zers are al kyl ci t rates, glycerol esters, al kyl pht hal ates, al kyl sebacates, sucrose esters, sorbi tan esters, di butyl sebacate and pol yet hyl ene gl ycol s 4000 to 20 000. Preferred plasticizers are tri butyl citrate, tri ethyl citrate, acetyl tri ethyl citrate, di butyl sebacate and di ethyl sebacate. The amounts used are between 1 and 35, preferably 2 to 10, % by weight, based on the ( met h) acryl ate copol ymer.
Optimizing Systemic Bi oavai I abi I i ty The amount of HX compound delivered by a solid medi cament composition i s substantially the same as that from an aqueous composition.
Administration of

- 44 -suf f i ci ent RB, as an ill ust rat i ye HX compound, to achi eve a ci rcul at i ng RB concent rat i on at the I C50 level woul d not by def i ni ti on be toxi c to al I
ci rcul at i ng I eukemi c cel Is ( i . e. , only approximately half of the I eukemi c cel I s woul d be affected at the I C50) . I n some embodi ments it can be preferred to admi ni ster RB i n an amount that is a multi pl e of the I C50 I evel , up to approxi mate! y 1500 mg ( i . e., 300 mM).
Alternatively, however, it can be suf f i ci ent to kill only a fraction of tumor cells as a result of an individual admi ni strati on.
The latter case can be preferable for avoi di ng a toxi c react i on ( i . e. , "tumor I ysi s syndrome") that can result from rapidly kill ed tumor cell burden. Thus, Howard et al . , N Engl J Med 364( 19) : 1844-1854 ( May 12, 2011) report that tumor I ysi s syndrome is the most common disease-related emergency encountered by physi ci ans treating hematol ogi c cancers such as I eukemi a.
As di scussed i n greater detail bel ow, it can al so be advantageous to kill only a port i on of the I eukemi c cells dun i ng a si ngl e treatment to i ni ti ate a f uncti onal i mmune response agai nst remai ni ng I eukemi a cell burden. A RB- i ni ti at ed functional immune system response i s believed to occur due at I east i n part from the act i on of RB-caused necrotic cell debris ci rcul at i ng i n the body i nduces an i mmune response that can prol ong the effects of an i ni ti al admi ni strati on of a hal ogenated xant hene such as RB.
An i nduced i mmune response can take a I onger ti me to develop than the more i mmedi ate kill i ng of the

- 45 -contacted I eukemi c cell s. That del ay i n effect can occur because of the ti me needed for i nducti on the appropri ate B and T cell popul at i ons to attack and kill the I eukemi c cell s as well as to i nduce I ong I asti ng memory T cells whose cont i nued ci rcul at i on can protect the patient from relapse. Such an initial del ay can be augmented for the subject's life-time due to the memory i mmune cell s so i nduced.
Combi nati on Treatment I n another aspect, an above pharmaceuti cal composi ti on is used i n conj unct i on with a second, differently-acting systemic cyt ot oxi c anti-leukemia agent; i . e. , a cytotoxi c ant i - I eukemi a agent whose mechanism of act i on i s different from that of the f i rst cytotoxi c agent, the HX compound. As noted previously, the hal ogenated xant henes I ocal i ze i n cancer cell I ysosomes, i ncrease the percentage of cell s in G1 phase of the cell cycle and i nduces cell death by apoptosi s [Swift et al . , Oncotargets Ther, 12:1293-1307 (February 2019)].
A first type of second anti-leukemia systemic cytotoxi c agent i s a so- cal I ed "small mol ecul e. " Such small mol ecul es can be vi ewed as semi - speci f i c cell ul ar poi sons i n that they are only general I y more specific at killing leukemia cells than non- I eukemi c cells.
Al most all small mol ecul e anti cancer agents are I ess leukemia-specific than a contemplated HX compound, and can result i n causi ng si ckness, baldness and other trauma to thei r reci pi ent subj ects that can lead to subj ects I eavi ng t hei r treatment regi mens.

- 46 -These small mol ecul es typi call y have molecular weights of about 150 to about 1000 Dal tons ( Da), and preferably about 250 to about 850 Da. Thi s group of small mol ecul es i nd l udes many of those used to treati ng hematol ogi c I eukemi as such as cal i cheami ci n (1368 Da), vi nbl ast i ne ( 811 Da), vi ncri sti ne ( 825 Da), i mat i nib ( 494 Da), monomet hyl auri st at i n (718 Da), et oposi de ( 589 Da), daunorubi ci n ( 528 Da), doxorubi ci n ( 544 Da), cl adri bi ne ( 286 Da), fl udarabi ne ( 365 Da), mi toxantrone ( 444 Da), 6- thi oguani ne ( 167 Da), met hot rexat e ( 454 Da), 6- mercaptopuri ne ( 152 Da), azacyt i di ne ( 244 Da), annamyci n (640 Da), soraf eni b (465 Da), cl of arabi ne ( 304 Da), ci spl at i n ( 300 Da), i ri not ecan ( 587 Da) and cytabari ne ( 243 Da) . One or more of the above small molecule anti-leukemia can comprise a second leukemia cytotoxi c agent. It is noted that many of these small mol ecul es are used as thei r sal ts, prodrugs and/or esters, whi ch consequently have greater molecular weights than those rounded val ues above.
A pharmaceuti cal composi ti on havi ng a second systemic cytotoxi c anti-leukemia agent can al so contain a small mol ecul e as above-described that is conj ugated to a I ager mol ecul e such as a protei n, detergent and/or pol ymer such as pol y( ethyl ene gl ycol ) [ PEG] . Such conj ugati ons of ten mi ni mi ze the toxi city of the small molecule and enhance si t us of delivery as use of an anti body that bi nds to a I eukemi c cell . Addi ti onal I y, a small mol ecul e cytotoxi c agent can be envel oped wi t hi n a Ii posome, mi cel I e or cycl odextri n mol ecul e that can be adapted to bi nd specifically bi nd to I eukemi c cells and/or be endocytosed by the leukemia

- 47 -cell . Thi s group of encapsul at ed and conj ugated small molecules i s i ncl uded with the previously di scussed small mol ecul e group of second systemi c cytotoxi c agents as t hei r act i ve cytotoxi c agent i s a small mol ecul e.
Ill ust rat i ve of such second systemic anti I eukemi a cytotoxi c agents are I i posomal daunorubi ci n, I i posomal annamyci n, sphi ngosomal vi ncri sti ne, I i posomal cytarabi ne, a cal i cheami ci n-conj ugated CD33 anti body cal led gemt uzumab ozogami ci n and a chi mer of CD30 anti body and monomethyl auri stati n E cal I ed brent uxi mab vedoti n.
Briefly, I i posomes are general I y spheri cal I y-shaped artificial vesi cl es typi cal I y prepared from chol est erol and phosphol i pi d mol ecul es that consti tute one or two bi layers and encapsul ate the smal I mol ecul e second systemi c cytotoxi c agent to assi st deli very.
See, Akbarzadeh et al . , Nanoscal e Res Lett, 8: 102 (2013) .
Cal i cheami ci n, i s a hi gh mol ecul ar wei ght small mol ecul e ( 1368 Da), and contai ns four I i nked sacchari des i nt err upted by a benzothi oate S- ester I i nkage as well as an ene- di yne group that cleaves DNA
sequences. Cal i cheami ci n is too toxi c to be used al one, LD50 i n nude mi ce of 320 [ig/ kg [ Di J oseph et al BI ood 103: 1807- 1814 ( 2004) ] . Si mi I ar I y, monomethyl auri stati n exhi bits general (broad range), hi gh toxi city [ I C50 < 1 nM for several cancer cell I i nes;
ApexBi o Technology Product Catalog ( 2013) ] that is mediated by linkage to an anti body against CD30 ( a TNF
receptor-family member that is a cell membrane protei n

- 48 -and cancer marker) was reported useful agai nst I arge cell I ymphoma and Hodgki n' s di sease [ Franci sco et al Blood 102: 1458- 1465 ( 2003) ], whereas linkage to an anti -CD79b monocl onal provi ded an advantage in treati ng three xenograft models of NHL [ Dornan et al . , Blood 114: 2721- 2729 (2009)1.
A systemic anti-leukemia medi cat i on that is a small molecule ( non- protei naceous, less than about 1000 grams/ mole) or a I arger protei naceous molecule, i s admi ni stered to the subj ect mammal to be treated such that the medication spreads throughout the subject's body. I nt ravenous admi ni strati on is one preferred method to achieve that spread of medication. On the other hand, i mat i ni b i s usual I y admi ni stered oral I y.
Illustrative small molecule anti-cancer medi cat i ons useful for treating I eukemi a include doxorubi ci n, et oposi de, vi ncri sti ne, ci spl at i n, i ri not ecan and cytarabi ne were used i n parental application Seri al No. 16/ 688, 319, whereas an exemplary protei naceous mol ecul e i s egasparagi nase. Of those small molecule medications, doxorubi ci n, et oposi de and vi ncri sti ne each of whi ch woul d be admi ni stered IV to a mammal i an subj ect appeared to synergi ze i n treatment with a sub-lethal dose of PV- 10 aqueous RB di sodi um sol uti on, and are preferred.
It is to be understood that admi ni strati on of any of the second I eukemi a systemi c cytotoxi c agents di scussed herei n can be undertaken multi pl e ti mes.
Such multi pl e admi ni strati ons are within the purvi ew of the treati ng physi ci an, and can be made i n conj uncti on with an admi ni strati on of the HX compound f i rst

- 49 -leukemia cytotoxi c agent or can be carried out separate! y.
A useful effective dosage of a small molecule systemic anti-leukemia medication is the dosage set out in the label i ng i nf ormat i on of a FDA-, nat i onal - or i nt er nat i onal agency- approved medi cat i on. Typi cal I y, monot herapy dose schedul es are set by determi ni ng the maxi mum tol erated dose ( MTD) i n early- stage cl i ni cal trials.
The MTD ( or a close variation thereon) is then promul gat ed to later-stage cl i ni cal trials for assessment eff i cacy and more detail ed assessment of safety. These MTDs frequently become the established t herapeut i c dose upon compl et i on of cl i ni cal test i ng.
However, because the small mol ecul e, systemi c anti -leukemia medi cat i on i s contempl at ed for use with an HX
compound i n a sol i d or I i qui d f ormul at i on, a MTD i s the maximal amount that would normally be used, and that amount is to be t i t rated downward f ol I owi ng usual procedures.
Exempl ary dosi ng schedul es for several systemic anti-cancer ( ant i - I eukemi a) medications ( agent s) t hat can be combi ned wit h hal ogenat ed xant hene therapy in the present invention are provided in Table A, below. It is noted that several of the medications I i sted bel ow are "smal I mol ecul es" as def i ned above, whereas others are large, protei naceous mol ecul es such as anti bodi es, preferably monoclonal anti bodi es, i nhi bit i nf I ammatory chemoki ne act i vi ty. They are nonet hel ess admi ni stered systemically.
The medi cat i ons of Table A are usually used as si ngl e active agents.
However, one or more can al so be used together, parti cul arl y the anti bodi es, as is the case with the

- 50 -immune checkpoi nt i nhi bi tor antibodies that are di scussed herei naf ter.
Tabl e A
Exempl ary systemi c i mmunomodul at ory or targeted anti cancer agents Systemic Agent Typical Dose Schedule adal i mumab 80 mg i ni ti al dose followed i n 1 week by 40 mg every other week SQ
brodal umab 210 mg subcutaneously (SC) at Weeks 0, 1, and 2, then 210 mg SC q2wk certol i zumab 400 mg initially and at weeks pegol 2 and 4 followed by 200 mg every other week or 400 mg Q4 weeks mai ntenance SQ
etanercept 50 mg twi ce weekly for 3 months followed by 50 mg once weekly SQ
gol i mumab 50 mg once a month SQ
gusel kumab 100 mg subcutaneous i nj ecti on once every 8 weeks, after starter doses at weeks 0 and i nf I i xi mab 5 mg/ kg given as an IV
i nduct i on regi men at 0, 2, and 6 weeks followed by a mai ntenance regi men of 5 mg/ kg every 8 weeks thereafter

- 51 -i xeki zumab 160 mg initial dose followed Q2 weeks with 80 mg unti I
week 12 then 80 mg Q4 weeks SQ
sari I umab 200 mg every 2 weeks as a subcutaneous i nj ect i on secuki numab 300 mg every week for 4 weeks then 300 mg every 4 weeks SQ
ust eki numab Less than 100 kg: 45 mg i ni ti ally, week 4 followed by 45 mg every 12 weeks SQ
More than 100 kg: 90 mg i ni ti ally, week 4 followed by 90 mg every 12 weeks SQ
apremi last Ti t rat ed dose over 5 days to work up to 30 mg twice daily PO
met hot r exat e Weekly single oral, IM or IV
to 25 mg per week or di vi ded 2. 5 mg dose at 12 hour i nterval s for three doses cycl ospori ne I ni ti al dose 2.5 mg/ kg/ day taken twice dai I y as divided ( BI D); dose t i t rated up to 4 mg/ kg/ day BID if response and I aboratory abnormal i ti es don' t ensue.
azat hi opri ne Used off label for ski n diseases, 1.0 mg/ kg oral or I V as a si ngl e dose or twi ce

- 52 -a day, dose maxi mum i s 2. 5 mg/ kg/ day.
Because of additive or synergistic effects, the combi nation therapy and method of treatment of the present i nventi on general I y permit use of the systemi c agent at a I evel at or bel ow the typi cal dose schedul e for the systemi c agent, such as those descri bed i n Table A, when used with an IV admi ni strati on therapy, such as that descri bed bel ow. However, the dosi ng schedules provided in Table A provide a useful guide for begi nni ng treatment from which dosages can be ti t rat ed to lessened amounts as seen appropriate by the physician caring for a given patient.
It is noted that a HX compound and a second cytotoxi c anti - I eukemi a agent need not be admi ni stered together nor by the same means of admi ni strati on.
Thus, a pi I I or capsul e form can be used for can be used to admi ni ster the HX compound f i rst cytotoxi c anti -1 eukemi a agent, whereas the small or 1 arge molecule second anti -1 eukemi a agent i s admi ni stered by IV or orally, like i mat i nib.
Those skilled in the art are aware of the van i ous met hods of admi ni steri ng anti -1 eukemi a agents.
A second type of second systemic cytotoxi c agent usef ul for combi nat i on t r eat ment wi th a hal ogenated xanthene such as that present i n aqueous RB
di sodi um sol uti on or a before-described solid dosage form i s an i mmune checkpoi nt i nhi bi tor, that can al so be viewed as a special systemic anti-leukemia medi cat i on. An i mmune checkpoi nt i nhi bi tor is a drug that bi nds to and bl ocks certai n checkpoi nt protei ns

- 53 -made by i mmune system cell s such as T cell s and some I eukemi a cell s. When not bl ocked, those protei ns i nhi bit i mmune responses, hel pi ng keep i mmune responses i n check and keepi ng T cell s or other immune cell s from kill i ng leukemia cells. BI ocki ng those immune checkpoi nt protei ns rel eases the "brakes" on the i mmune system permitting immune cells to become activated and kill I eukemi a cell s.
A useful i mmune checkpoi nt i nhi bi tor i s preferably a human or humani zed monocl onal anti body or bi ndi ng portion thereof whose admi ni strati on blocks the act i on of those certai n protei ns. That blockage permits the immune system to recognize the leukemia cell s as foreign and assi St i n el i mi nati ng those I eukemi a cell s from the body.
Ill ust rat i ve i mmune checkpoi nt inhibitors include the ant i - CTLA- 4 ( cyt ot oxi c T I ymphocyt e-associ at ed anti gen 4) monocl onal anti bodi es i pi I i mumab and t remel i mumab that are desi gned to counter down-regulation of the immune system by blocking CTLA- 4 activity and thus augment T cel I response agai nst I eukemi a.
Si mi I ar I y, monocl onal anti bodi es such as pi di I i zumab, ni vol umab, ti sl el i zumab, spart al i zumab, cemi pl i mab, pembrol i zumab, camr el i zumab, si nti I i mab, tor i pal i mab, and dostar I i mab bi nd to PD-1 ( programmed death 1) receptor to counter down- regul at i on of the i mmune system and augment T cel I responses to cancerous cells.
Three monocl onal antibodies that target the immune checkpoi nt protein I i gand (anti - PD- L1) for the PD-1 receptor ( ant i - PD- 1) are at ezol i zumab, avel umab, and durval umab. I ni ti al work with anti bodi es to the PD-1 receptor I i gands, PD- Li and PD- L2, such as BMS-

- 54 -936559 and MEDI 4736 ( durval umab) to PD-Li, al so i ndi cat e i nhi bi ti on of down- regul at i on of the i mmune system and an augmented T cell response agai nst I eukemi a.
The above i mmune checkpoi nt i nhi bi tor anti bodi es have been found useful when admi ni stered si ngl y al ong with a HX compound as well as usi ng two different types of i mmune checkpoi nt i nhi bi tors al ong with an HX compound. A poster by Patel et al AMERICAN SOCIETY of CLINICAL ONCOLOGY ( ASCO) 2020 VI RTUAL SCI ENTI Fl C PROGRAM May 29- 31, 2020 provi ded data from a study that utilized rose bengal di sodi um that was i nj ected i nt rat umor al I y i nt o uveal mel anoma tumors metastati c to the liver al ong with either a systematic admi ni strati on of anti - PD-i, or systemi c admi ni strati ons of both anti - PD- 1 and CTLA- 4 anti bodi es.
More recently, several further groups of anti bodi es with checkpoi nt i nhi bi tor act i vi ty have been identified, and because of t hei r similarity of act i on, are deemed herei n to be i mmune checkpoi nt i nhi bi tors.
One ill ust rat i ve group i mmunoreact with the cell surf ace receptor 0X40 (CD134) to stimulate prol i f erat i on of memory and effector T- I ymphocytes, and thereby st i mul ate a T- cel I - medi at ed immune response agai nst cancerous cel I s. Exempl ary such humani zed anti - 0X40 monocl onal anti bodi es i ncl ude those presently referred to i n the literature as gsk3174998 ( I gG1), pogal i zumab (MOXR0916), MED10562 and the human ant i -OX40 I gG2 antibody designated PF- 04518600 ( PF- 8600) .
Another group i mmunoreacts with lymphocyte act i vat i on gene 3 prot ei n ( ant i - LAG- 3; CD223) t hat

- 55 -negatively regul at es T lymphocytes by bi ndi ng to the ext race! I ul ar domai n of the I i gand, thus avoi di ng autoi mmuni ty caused by T cell overact i vat i on.
LAG-3 is an i mport ant i mmune checkpoi nt in vi vo and pl ays a bal anced regul at ory rol e in the human i mmune system [ Shan et al . , Oncol Lett 20: 207 ( 2020)1.
The molecule LAG-3 blocks the signal transduct i on pathway of T cell act i vat i on; however, the intracellular segment of the LAG-3 molecule produces i mmunosuppressi ve signals, which have been found to regul ate CD4+T cell activity.
LAG-3 regul at es the immune response of T cel Is in three ways: Fi rst, it di rect I y i nhi bits the proliferation and activation of T
cell s vi a negative regul at i on of T cells. Second, it can promote the i nhi bi tory function of regul at ory T
cells (Tregs), and the T cel I response can then be indirectly inhibited.
Thi rd, it can prevent T cell activation by regul at i ng the function of antigen presenting cells ( APCs) [ J ol I er et al . , Curr Top Mi crobi ol I mmunol 410: 127-156 ( 2017) ] .
To date, no monocl onal anti body to LAG-3 is known to be been approved for sale and use by the US
Food and Drug Admi ni strati on.
Studies are ongoi ng usi ng a humani zed I gG4 monocl onal anti body from Merck ref erred to as MK-4280, and another from Bristol Myers Squi bb with the I NN name rel at I i mab.
A still further type of immune checkpoi nt i nhi bi tor i s a monocl onal anti body agai nst CD47 and macrophage checkpoi nt i nhi bi tor that i nterf eres with recognition of CD47 by the SIRPa receptor on macrophages, thus bl ocki ng the "don' t eat me" signal used by cancer cel Is to avoi d bei ng i ngested by

- 56 -macrophages. Thi s monocl onal , whose I NN name i s magrol i mab, i s bei ng devel oped by Gil i ad Sci ences, I nc.
i n several hematol ogi c and sol i d tumor mal i gnanci es, i ncl udi ng myel odyspl ast i c syndrome ( MDS) .
Magrol i mab has been granted Fast Track Desi gnat i on by the FDA for the treatment of myel odyspl ast i c syndrome ( MDS), acute myel oi d I eukemi a ( AML), diffuse I arge B- cell I ymphoma ( DLBCL) and f ol I i cul ar I ymphoma.
Magrol i mab has al so been granted Orphan Drug Designation by the FDA for MDS
and AML and by the European Medicines Agency for AML.
Monocl onal anti bodies to T cell i mmunogl obul in and muci n domain 3 (anti -TI M-3), another checkpoi nt marker, are i n early devel opment by Novart i s Oncol ogy Co. under the I NN name sabatol i mab ( earl i er MBG- 453) for use i n MDS and AML therapy based on di f f erent i al I eukemi c stem cell expressi on, its role as a co- i nhi bi tory T- cell co- receptor, and possi bl y a rol e in promot i ng anti body-dependent cellular phagocyt osi s (ADCP) . TI M- 3 is expressed on AML leukemic progenitors but i s not seen on normal hematopoi et i c stem cell s and its expression has been correlated with severity of myel odyspl ast i c syndromes as well as the I i kel i hood of progression to AML. There are currently multiple trials of TI M-3 anti body MBG- 453 in f ront I i ne myel odyspl ast i c syndromes and AML, with encouraging ant i I eukemi c activity presented when used in combi nat i on with deci tabi ne.
I nt act monocl onal anti bodi es, as well t hei r paratope- contai ni ng port i ons (binding site-containing port i ons) such as Fab, Fab' , F( ab' )2 and Fv regi ons, as well as si ngl e- stranded pept i de bi ndi ng sequences can be useful as i mmune checkpoi nt protei n i nhi bi tors.

- 57 -Most of these anti bodi es are admi ni stered vi a an IV
route. I nt act checkpoi nt i nhi bi ti ng monocl onal anti bodi es have half-lives i n a human body of about one to three weeks [ e. g. , Yervoye ( i pi I i mumab) t ermi nal t 1/ 2 = 15.4 days; package insert 12/2013; Keyt ruda() ( pembrol i zumab) termi nal t 1/ 2 = 23 days; package i nsert 03/201]], and single-stranded ol i go or pol ypept i des tend to have shorter hal f - I i ves in vivo.
Because of the relatively short half-lives of the smal I mol ecul e second cytotoxi c anti - leukemia agents and a hal ogenated xanthene compound- contai ni ng medi cament, both medi caments can be admi ni stered i n a si ngl e composition or i n separate composi ti ons. If admi ni stered separately, it is preferred to admi ni ster both types of anti - cancer (anti - leukemia) agent wi t hi n mi nut es to about 3 hours of each other. More preferably, both are admi ni stered wi t hi n less than one hour of the other.
The word ''admi ni strati on" i s used herei n to mean the beginning of a treatment regimen. Thus, swal I owi ng a tablet or other per os dosage form i s the begi nni ng of a treatment regi men, as i s the ti me at which an IV fl ow is begun. When both first and second cytotoxi c anti -I eukemi a agents are present together i n the same, si ngl e composi ti on, admi ni strati on begi ns when that unitary composi ti on enters the subject's body.
Where the second cytotoxi c systemi c anti -1 eukemi a agent i s an i mmune checkpoi nt i nhi bi tor such as a monocl onal anti body, the hal ogenated xant hene compound and the second cytotoxi c anti-leukemia agent

- 58 -i mmune checkpoi nt i nhi bi tor can be admi ni stered together or one before the other, with the second cytotoxi c anti I eukemi a agent i mmune checkpoi nt i nhi bi tor bei ng admi ni stered up to about one month prior to the hal ogenated xanthene.
Preferably, the two cytotoxi c ant i - I eukemi a agents are admi ni stered together or with the second systemic cytotoxi c anti -I eukemi a agent i mmune checkpoi nt i nhi bi tor bei ng admi ni stered wi t hi n a few days after the hal ogenated xant hene. A second systemic cyt ot oxi c anti-leukemia agent i mmune checkpoi nt i nhi bi tor can al so be admi ni stered about one month after the hal ogenated xant hene.
Studi es Mi ce:
Female C17 SCI D mice from Charles River Cell I i ne used in xenograf t :
The SEM cel I I i ne was i ni ti ally established from a 5 year old female with B acute I ymphobl ast i c leukemia.
It carries the MLL-AFFF1 gene fusion and heterozygous for CDKN2A and TP53.
The utility of this cell I i ne i n anti -cancer drug sensitivity studies has been descri bed ( Bar ret i na et al . Nature 483: 603- 607, 2012) .
2.5x106 exponentially growing SEM cells [ I abel led with green fl uorescent protei n (GFP) ] were injected i nt ravenous! y i nto each ani mal and the establishment of tumors was monitored. After 4 weeks to permit the growth of the tumors, mice were randomized to three groups.
Group 1 ( n=9). Control :

- 59 -These ani mal s recei ved 100 ilL of PBS given oral I y twi ce a week for two weeks.
Group 2 ( n=8). Treatment Cohort I :
These animals received 25 1.11_ of PV- 10 (bob rose bengal di sodi um w/v i n 0. 9 percent i n aqueous sal i ne) di I uted i n PBS to a f i nal vol ume of 100 111_ and given oral I y by gavage twi ce a week for 2 weeks.
Group 3 ( n=8). Treatment Cohort I I .
These animals received 12.5 tiL of PV- 100 ( as discussed above) di I uted i n PBS to a final vol ume of 100 !IL and given oral I y by gavage twi ce a week for 2 weeks.
Evi dence of di sease progressi on was moni tored i n al I ani mal s and survi val was f ol I owed up to 120 days following the i ni ti at i on of treatment. Data are presented i n Fig. 1 as Kapl an- Mei er esti mates.
As is seen from the graphs of Fig. 1, oral delivery of the HX compound is clearly evidenced by dose-dependent survival of the treated mice.
The results di scl osed i n parental appl i cat i on Seri al No. 16/688, 319 showed that el even commerci ally avail abl e I eukemi a cell I i nes der i ved from pat i ents with either pri mary or relapsed pedi at ric I eukemi a that were treated with PV- 100 and two primary leukemia samples in cell culture could be killed by admi ni strati on of i ncreasi ng doses of PV- 10 . Cell vi abi I i ty was measured by al amar bl ue assay, 96 hours post- t r eat ment .
PV- 100 admi ni strati on decreased I eukemi a cell vi abi I i ty i n a concent rat i on and ti me dependent manner in the el even pediatric leukemia cell lines ( mean 1050

- 60 -92.8 M), and three primary leukemia samples ( mean I C50 122. 5 M) exami ned. The resul ts show that PV- 10 i s cytotoxi c to I eukemi a cell I i nes with a mean I C50 val ue of 92.8 124 (Table 1, below) and is cytotoxic to two primary leukemia samples with a mean I C50 value of 122.5 p.M (Table 2, below) .
Table 1*
Cell Line Cell Type PV-10 I C50 PM
KOPN8 I nf ant ALL 150 CEM- Cl T- ALL 121 Kas umi 1 AML 72 MV4- 11 Bi phenot ypi c 68 Mol m13 AML 42 Mol t 4 1-ALL 41 Mol t 3 1-ALL 35 Mean 92.8 *Hal f maxi mal i nhi bi tory concentration (1 C50) val ues f or pedi at ri c I eukemi a cel I I i nes treated wi th PV- 10 for 96 hours.
Tabl e 2**
Cell Type PV-10 1050 pm Infant AML 95 Mean 122.5 **Hal f maxi mal i nhi bi t ory concent r at i on ( I C50) val ues for pri nary pedi at ri c I eukemi a sampl es treated with PV- 10 for 96 hours.

- 61 -Si mi I ar resul ts were separately obtai ned usi ng I eukemi a cel I I i nes CCRF- CEM, HL- 60( TB) , K- 562, MOLT-4, RPM! - 8226 and SR.
It was surpri si ng that I eukemi a cell s coul d be killed at all at the concentrations of RB di sodi urn used i n these studi es, which come out to approximately 10- 4 to about 10- 5 molar based on the I C50 values.
Even so, the concent rat i ons of HX compound requi red were greater than those bel i eyed achi eyed by oral admi ni strati on i n the present studies as was discussed previ ousl y. Thus, it was st i I I more surpri sing that

62.5 % of the HX Compound-treated leukemic mice survived for 120 days whereas the untreated leukemic mice and those treated with lesser amounts of HX
compound exhi bi ted much poorer survi val rates as are shown by the data i n Fi g. 1.
Each of the patents, patent applications and art i cl es cited herei n is i ncorporated by reference.
The arti cl es "a" and "an" are used herei n to refer to one or to more than one ( i . e., to at I east one) of the grammatical object of the article. Each of the patents, patent appl i cat i ons and art i cl es cited herei n i s i ncorporated by reference.
The f oregoi ng descri pti on and the exampl es are intended as illustrative and are not to be taken as I i mi ti ng. St i I I other van i at i ons within the spi ri t and scope of t hi s i nventi on are possi bl e and will readily present themselves to those ski I I ed i n the art.

Claims (30)

CLAI MS:

1. A met hod of t reat i ng a mammal i an subj ect havi ng l eukemi c cel l s compri si ng the step of oral l y admi ni steri ng a t herapeuti cal l y eff ecti ve amount of a hal ogenated xanthene, a pharmaceuti cal l y accept abl e sal t, a l actone, or a C1- C4 al kyl or aromati c ester thereof ( HX compound) as a f i rst l eukemi a cytotoxi c agent di ssol ved or di spersed i n a pharmaceuti cal I y accept abl e sol i d or I i qui d composi ti on to sai d mammal i an subj ect havi ng l eukemi c cel l s.

2. The method accordi ng to cl ai m 1, wherei n sai d oral admi ni strati on i s repeated.

3. The method accordi ng to cl ai m 1, wherei n sai d composi ti on i s a sol i d.

4. The method accordi ng to cl ai m 3, wherei n the sai d HX compound i s di ssol ved i n or di spersed i n or on a sol i d di l uent medi um.

5. The method accordi ng to cl ai m 3, wherei n the sol i d composi ti on is in the f orm of a tabl et, l ozenge, or a pl ural i ty of general l y spheri cal sugar pri l I s.

6. The method accordi ng to cl ai m 5, wherei n sai d sol i d composi ti on i s compri sed of general I y spheri cal sugar pri l l s coated wi th one or a pl ural i ty of l ayers of sai d HX compound.

7. The method accordi ng to cl ai m 3, wherei n sai d sol i d composi t i on i s coat ed wi t h a bar r i er fi I m that reduces the rate of di ssol uti on and/or di si ntegrati on of the composi ti on i n aqueous medi a.

8. The method accordi ng to cl ai m 7, wherei n sai d barri er film coati ng i s an enter i c coati ng that di ssol ves and/or di si nt egr at es at a physi ol ogi cal pH
val ue of 5 or greater.

9. The method accordi ng to cl ai m 1, wherei n sai d f i rst cancer cytotoxi c agent hal ogenated xanthene compound i s rose bengal , a pharmaceuti cal l y acceptabl e sal t, a l actone, or a C1- C4. al kyl or aromati c ester t hereof .

10. The method accordi ng to cl ai m 1, wherei n sai d HX compound i s rose bengal , a pharmaceuti cal l y accept abl e sal t, a l act one, or a C1- C4 al kyl or aromat i c est er t hereof .

11. The method accordi ng to cl ai m 1, wherei n sai d composi ti on i s an aqueous I i qui d.

12. The method accordi ng to cl ai m 11, wherei n sai d aqueous I i qui d composi ti on i s as an osmol al i ty that i s l ess than that of normal human osmol al i ty.

13. The method accordi ng to cl ai m 1, wherei n sai d admi ni strati on step i s car r i ed out i n conj uncti on wi t h admi ni st rat i on to sai d mammal i an subj ect of a second therapeuti cal I y eff ecti ve amount of a second, di f f erent I y- act i ng syst emi c l eukemi a cyt ot oxi c agent di ssol ved or di spersed i n a pharmaceut i cal l y accept abl e medi um, wherei n sai d systemi c l eukemi a cytotoxi c agent i s a smal l mol ecul e, a protei naceous mol ecul e t hat i nhi bi ts i nf I ammatory chemoki ne act i vi ty, i oni zi ng radi at i on t herapy and i nt act checkpoi nt i nhi bi tor anti bodi es or paratope- contai ni ng port i ons thereof. .

14. The method accordi ng to cl ai m 13, wherei n sai d second l eukemi a cytotoxi c agent i s di ssol ved or di spersed i n a pharmaceut i cal l y accept abl e sol i d medi um.

15. The method accordi ng to cl ai m 14, wherei n t he pharmaceuti cal l y accept abl e sol i d medi um contai ni ng t he second l eukemi a cytotoxi c agent i s admi ni stered per os.

16. The method accordi ng to cl ai m 13, wherei n second l eukemi a cytotoxi c agent i s i oni zi ng radi at i on.

17. The method accordi ng to cl ai m 13, wherei n sai d smal l mol ecul e exhi bi ts synergy wi t h sai d f i rst l eukemi a cytotoxi c agent .

18. The method accordi ng to cl ai m 13, wherei n sai d second l eukemi a cytotoxi c agent i s di ssol ved or di spersed i n a pharmaceut i cal l y accept abl e aqueous medi um.

19. The method accordi ng to cl ai m 18, wherei n the pharmaceuti cal l y accept abl e aqueous medi um contai ni ng t he second l eukemi a cytotoxi c agent i s admi ni stered i nt ravenous! y.

20. The method accordi ng to cl ai m 13, wherei n second l eukemi a cytotoxi c agent i s a smal l mol ecul e havi ng a mol ecul ar wei ght of about 150 to about 1000 Da.

21. The method accordi ng to cl ai m 20, wherei n sai d smal l mol ecul e i s sel ected f rom one or more of the group consi sti ng of vi nbl asti ne, vi ncri sti ne, i mat i ni b, monomethyl auri st at i n, et oposi de, daunorubi ci n, doxorubi ci n, cl adri bi ne, f I udar abi ne, mi t oxant r one, 6- t hi oguani ne, met hot r exat e, 6- mercaptopuri ne, azacyt i di ne, annamyci n, soraf eni b, cl of arabi ne, ci spl at i n, i ri not ecan and cytabari ne.

22. The method accordi ng to cl ai m 19, wherei n the second l eukemi a cytotoxi c agent compri ses i nt act monocl onal anti bodi es or paratope- contai ni ng port i ons t hereof .

23. The method accordi ng to cl ai m 22, wherei n sai d i ntact monocl onal anti bodi es or paratope-contai ni ng port i ons thereof are i mmune checkpoi nt i nhi bi tors.

24. The method accordi ng to cl ai m 23, wherei n sai d i mmune checkpoi nt i nhi bi tors bi nd to one or more protei naceous materi al s sel ected f rom one or more of t he group consi st i ng of ant i - CTLA- 4, ant i - PD- 1, ant i - PD- Ll, ant i - PD- L2, ant i - OX40, ant i - LAG- 3, ant i -CD47 and anti - TI M- 3.

25. The method accordi ng to cl ai m 22, wherei n sai d i nt act monocl onal anti bodi es are protei naceous mol ecul es t hat i nhi bi t i nf I ammatory chemoki ne act i vi ty that are sel ected f rom the group consi sti ng of adal i mumab, brodal umab, certol i zumab pegol , et aner cept , gol i mumab, gusel kumab, i nf I i xi mab, i xeki zumab, sari I umab, secuki numab, and usteki numab.

26. The method accordi ng to cl ai m 19, wherei n sai d anti bodi es or par at ope- contai ni ng port i ons thereof are admi ni stered after admi ni strati on of sai d HX compound.

27. The method accordi ng to cl ai m 19, wherei n sai d anti bodi es or par at ope- contai ni ng port i ons thereof are admi ni stered bef ore admi ni st rat i on of sai d HX compound.

28. The method accordi ng to cl ai m 19, wherei n sai d anti bodi es or par at ope- contai ni ng port i ons t hereof are admi ni stered concurrentl y wi t h admi ni strati on of sai d HX compound.

29. The method accordi ng to cl ai m 19, wherei n sai d f i rst HX compound and sai d second I eukemi a cytotoxi c agents are admi ni stered si mul taneousl y to wi t hi n about 3 hours of each other.

30. The method accordi ng to cl ai m 13, wherei n sai d HX compound i s rose bengal , a pharmaceuti cal l y acceptabl e sal t, a l actone, or a C1- C4 al kyl or aromati c ester thereof. .