Liposomal daunorubicin: in vitro and in vivo efficacy in multiple myeloma

HEMATOLOGICAL ONCOLOGYHematol. Oncol. 16: 47–55 (1998)

LIPOSOMAL DAUNORUBICIN: IN VITRO AND IN VIVOEFFICACY IN MULTIPLE MYELOMA

. , . . , . . , . . , . . . , . . ,. . , . . . . *

Department of Haematology, The General Infirmary at Leeds, Great George Street, Leeds, U.K.

SUMMARY

Liposomal encapsulation of anthracyclines is a potential method of drug targeting, altering both theantitumour activity and side-effect profile of anthracyclines. Liposomal daunorubicin (daunoxome) showsboth altered pharmacokinetics and a potential for reducing dose-limiting cardiotoxicity compared toconventional daunorubicin. Anthracyclines have a common role in the treatment of multiple myeloma, aprevalent and fatal haematological malignancy. Avoiding cumulative anthracycline toxicity in thesepatients is important. There is also a need for more effective relapse schedules given that many patientshave chemosensitive disease at relapse. We have analysed daunoxome in vitro in myeloma cell lines usinga thymidine-based cytotoxicity assay and show superior efficacy compared to a pegylated liposomaldoxorubicin derivative. Subsequently we have treated seven relapsed myeloma patients with a regimeconsisting of oral CCNU 25–50 mg/m2 on day 1, 4 days of oral dexamethasone 10 mg/m2 and intravenousdaunoxome (liposomal daunorubicin) given for 4 days (total 100 mg/m2). The main toxicity wasmyelosuppression but non-haematological toxicity was minimal and the regime was well tolerated. Fourout of seven of these heavily pretreated patients responded. Together with the in vitro data onits cytotoxicity in myeloma and its favourable pharmacokinetic profile further studies of liposomaldaunorubicin in myeloma would be warranted. Copyright ? 1998 John Wiley & Sons, Ltd.

daunorubicin; doxorubicin; liposomes; multiple myeloma

INTRODUCTION

Anthracycline antibiotics, including daunorubicin and doxorubicin, are a family of antineo-plastic agents widely used in the treatment of haematological and solid malignancies.1,2 Cardio-toxicity is a unique characteristic of these agents, related to the total cumulative dose, and isgenerally irreversible and refractory to medical therapy.1,2 Liposomal encapsulation alters boththe pharmacodynamic and pharmacokinetic properties of drugs and liposomal composition andsize are important in determining these effects.3–5 An advantage of liposomal anthracyclineformulations is the potential for reductions in observed dose-limiting cardiotoxicity relative toeither daunorubicin or doxorubicin as free drug.6–8 Liposomal formulations will also alter theantitumour activity of the encapsulated drug both as a consequence of alteration of pharma-cokinetic and pharmacodynamic properties. Compared to conventional daunorubicin, thecurrently commercially available form of liposomal daunorubicin (daunoxome) has a longerinitial half-life and a 200- to 400-fold reduction in the volume of distribution indicating itsintravascular distribution giving both higher peak plasma levels and a larger AUC.7,9 For an

*Correspondence to: Dr G. J. Morgan, Department of Haematology, University of Leeds, The General Infirmary atLeeds, Great George Street, Leeds LS1 3EX, U.K.

CCC 0278–0232/98/020047–09$17.50 Received 21 July 1998Copyright ? 1998 John Wiley & Sons, Ltd. Accepted 30 September 1998

48 . .

80 mg/m2 dose the peak levels, initial half-life and AUC for daunoxome compared to freedaunorubicin are 43·67 versus 0·4 ìg/ml, 5·2 versus 0·77 h, and 375·3 versus 10·33 (ng/h/ml).Animal studies support the conclusion that liposomal daunorubicin delivery operates throughtargeting in tandem with the sustained plasma levels10 and preclinical animal studies confirmedincreased in vivo delivery of daunorubicin to solid tumours by up to 10-fold compared toconventional daunorubicin.11 Liposomal encapsulation (with diameters<100 nm) also enhancesdrug delivery to normal tissues with sinusoids/fenestrated capillaries12 such as bone marrow,liver and spleen, and increases the effective marrow dose which is a particular advantage forhaemopoietic neoplasms based in the marrow. Liposomal daunorubicin has been shown to havecomparable efficacy to previous treatments in advanced AIDS-related Karposi’s sarcoma with asignificantly improved safety profile.13,14 The two other liposomal anthracycline formulationscontain doxorubicin and differ from the daunorubicin formulation both in increased size(approximately daunoxome 45 nm, doxil 100 nm and D99 180 nm) and, in the case of doxil, thepresence of a methoxypolyethylene glycol coating (pegylation) on the liposomal exterior in anattempt to prolong the circulating half-life of the anthracycline. D-99 is still being evaluated,prior to licensing and is not yet commercially available.

Comparison of these formulations with their unencapsulated generic compounds inhaemopoietic malignancies is therefore worthwhile.

Multiple myeloma remains an incurable malignancy with a median survival of only 30months. Myeloma has an incidence of 10 in 100 000 at the age of 60 years making it one of thecommonest haematological malignancies for this age group. Survival from relapse is long witha median survival of 18 months15 and so there is a need for effective relapse schedules.Anthracyclines are commonly used in multiple myeloma being incorporated into a number ofwell-established regimes. Often relapsing patients have received prior anthracyclines in regimessuch as ABCM and C-VAMP so avoiding toxicity related to this is important. We have assessedthe comparative cytotoxicity of daunorubicin and doxorubicin relative to two commerciallyavailable liposomal derivatives in vitro using multiple myeloma cell lines. Subsequently we havegone on to treat seven patients with relapsed multiple myeloma with a regime which is amodified version of the CIDEX regime16 using liposomal daunorubicin rather than conventionalanthracycline together with an alkylating agent and oral steroid with the aim of evaluating theefficacy and safety profile of such a regime. The regime consists of oral CCNU 25–50 mg/m2 onday 1, 4 days of oral dexamethasone 10 mg/m2 and intravenous daunoxome (liposomaldaunorubicin) given for 4 days (total 100 mg/m2). Several of the most popular chemotherapyregimes17,18 used in myeloma such as VAD and C-VAMP use a continuous infusion ofanthracycline over several days in order to kill cells which go into cycle. The pharmacokineticprofile of daunoxome provides sustained plasma levels following a short infusion and thus offersa practical alternative to continuous infusion of conventional anthracyclines. The plannedinterval between courses was 21–28 days and the dose of daunoxome used was similar to thatused in previous studies of daunoxome in NHL.19,20

METHODS

Cell linesFour cell lines were assessed namely two myeloma cell lines (U266 and JJN3), a glioblastomacell line (U251) and a histiocytic lymphoma cell line (U937). The method used measuredthymidine uptake, measuring the ability of drugs to inhibit cell proliferation and was based onthe method used by Wiles et al.21 which examined a range of human tumour cell lines (e.g.breast, lung, ovarian, prostate, melanoma, brain, lymphoma and leukaemia tumour cell types).

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All cell lines were maintained in RPMI medium supplemented with 10 per cent fetal bovineserum at 37)C with 5 per cent CO2 in standard humidified tissue culture incubators and each celltype was passaged twice a week, the U251 cell line being adherent required trypsinization.

For the cell proliferation inhibitor assay, cells were harvested, pelleted by centrifugation andresuspended in supplemented media at a concentration of 105/ml and dispersed into 96-welltissue culture plates at a seeding density of 1#104 cell per well in 100 ìl aliquots. Viable cellcounts were determined using a haemocytometer and trypan blue exclusion. Following seedingthe adherent cell line U251 was maintained for 4 h under tissue culture conditions to allow cellattachment which was confirmed microscopically.

Stock solutions of daunorubicin, doxorubicin and two commercially available liposomalforms, namely daunoxome (liposomal daunorubicin) and doxil (pegylated liposomal doxo-rubicin) were diluted into appropriate supplemented media and serial 10-fold dilutions of eachprepared in 96-well plates. Aliquots (100 ìl) of each concentration of drug were then added tothe corresponding well of the 96-well tumour plate to give final concentrations of drug rangingfrom 105–10"3 ng/ml in a total volume of 200 ìl per well with four wells for each concentrationof drug. Positive controls received 100 ìl of supplemented medium alone whilst negativecontrols also received 20 ìl of TRIS (trishydroxymethylaminomethane) base as an inhibitor ofcell proliferation.

Finally 0·25 ìCi of methyl-[3H]-thymidine was added to each well (125 ìl of 1 mCi/ml stockwas added to 10 ml of minimal essential media (MEM), filtered through a 0·2 ìm filter and 20 ìlwas added to each well of each plate).

Cells were then incubated under tissue culture conditions for 24 h for the U266, JJN3 andU251 cell lines and 42 h for the U937 cell line (assay duration was calculated to be greater thanthe length of one doubling time). Following incubation cells were lysed by addition of sodiumhydroxide and lysed cells were harvested using a semi-automated cell harvester onto a glass fibrefilter mat with repeated washing steps to remove unincorporated methyl-[3H]-thymidine. Filtermats were then air dried for at least 12 h. Each disc of filter mat (corresponding to the harvestedcell lysate from a single well) was punched out into a scintillation vial to which was added 1 mlof scintillation fluid. The vials were then transferred to a scintillation counter to determinec.p.m. 3H. All data were analysed using Prism 2.01, GraphPad Software, which after normali-zation of data, fitted data to a standard four-parameter nonlinear regression equation andcalculated 50 per cent inhibitory conditions for each treatment condition (IC50). Significance ofdata was determined by Student’s t-test (unadjusted for multiplicity of comparison; conservativedegrees of freedom) with significance accepted for p<0·05.

Chemotherapy regime

Oral CCNU 25–50 mg/m2 day 1 only, oral dexamethasone 10 mg/m2 days 1–4, intravenousdaunoxome (liposomal daunorubicin) 40 mg/m2 day 1 and 20 mg/m2 days 2–4.

Patients

We have treated seven patients with multiple myeloma with the above regime based on thecommonly used CIDEX regime.16 The characteristics of the patients are shown in Table 1. Themedian age of the patients was 52 years (49–69 years). All seven patients had advanced diseasewith a median time of 49 months (20–120 months) since diagnosis. Two patients had relapsedafter two peripheral blood stem cell transplants (PBSCT), three after a single PBSCT and theother two patients had both received three types of chemotherapy and were ineligible for aPBSCT because of age (one patient) or failure to mobilize stem cells (one patient). All seven

Copyright ? 1998 John Wiley & Sons, Ltd. Hematol. Oncol. 16: 47–55 (1998)

Table 1. Characteristics of patients treated with CDD regime and their response

Age atfirst Time since

Totalprevious

e Paraproteinlight chain Plasma cells Clinical response

No change >50%reduction

Symptomaticimprovement.Relapse 3 months

Light chaindecrease by64%

>50%reduction

Symptomaticimprovement.Relapse 3 months

No change Not assessed Died 16/1/98 ofprogressivedisease. Onlyreceived 1 course

70%reduction

From 58%to<5%

Symptomaticimprovement.Proceeded tointermediate dosemelphelan

No change Proceeded toalternativetherapy

Notassessed

Not assessed Died ofgastrointestinalbleed

Light chaindecrease by19%

>50%reduction

Further twocourses

50

.

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PatientCDD

(years)diagnosis(months) Previous treatment

Echocardiogramprior to CDD

anthracyclindose

PC 49 49 C-VAMP#4, PBSCT No. 1 5/96(HDM), a-interferon, relapse 1/96,C-VAMP#4, CIDEX#3, PBSCTNo. 2 11/96 (BEAM), relapse 6/97

Normal 288 mg/m2

adr,90 mg/m2

idarJL 50 67 ABCM#8, a-interferon, relapse

3/94, C-VAMP#6, PBSCT No. 1(HDM), relapse 11/95, Z-DEX#8,PBSCT No. 2 (cyclo-TBI), relapse6/97

Normal 456 mg/m2

adr,330 mg/m2

idar

PT 69 120 ABCM, C weekly and intermittentoral melphalan with stable disease.Past history of ischaemic heartdisease, chronic renal failure

Impaired 44%EF

300 mg/m2

adr

JB 49 20 ABCM#7, disease progressed 1/97.Vinorelbine#3, CIDEX#4 but noresponse

ImpairedEF>50% EF

210 mg/m2

adr,120 mg/m2

idar

LP 52 31 Plasmacytoma of spine 10/91, txradiotherapy, Myeloma 4/95,C-VAMP#5, PBSCT (HDM),a-interferon 1/96–7/97, relapse,Z-DEX#3 but no response

Very impaired,19% EF

180 mg/m2

adr,120 mg/m2

idar

EE 63 58 ABCM#4, stable disease.Hyperparathyroidism, 3#C-VAMP,oral cyclo, PBSCT (HDM) 28/3/96.Lytic lesion R forearm 10/97

Normal 227 mg/m2

adr

RJ 52 31 Light chain myeloma, C-VAMP#5,PBSCT (HDM) 1/96, a-interferonuntil 11/97, relapse with increasedlight chain excretion

Normal 180 mg/m2

adr

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patients had previously received adriamycin at a median cumulative dose of 227 mg (180–456 mg) and four patients had also received idarubicin at a median cumulative dose of 120 mg(90–330 mg). Echocardiography was abnormal prior to chemotherapy in three patients withreduced ejection fractions of 50, 44 and 19 per cent. None of these patients had clinical heartfailure.

The number of courses received varied: four courses (one patient), three (two patients), two(three patients) and one (one patient). The median interval between courses was 31·5 days(21–55 days). The planned interval between courses was 21–28 days.

RESULTS

In vitro analysis

For the four cell lines analysed our data indicate that daunorubicin, doxorubicin and liposomaldaunorubicin are substantially more cytotoxic than the commercial liposomal preparation ofdoxorubicin. Conventional anthracyclines are also more cytotoxic than liposomal daunorubicinpresumably as a consequence of liposomal encapsulation effecting drug release during the timeof the assay. For the myeloma cell line U266B the IC50 values (ng/ml) for daunorubicin,doxorubicin, liposomal daunorubicin and liposomal doxorubicin were 1·37, 4·19, 80·5 and28 500. The respective figures for the myeloma cell line JJN3 were 5·6#10"2, 0·24, 7·25 and573·7. Table 2 summarizes the four cell lines in terms of IC50 and 95 per cent confidenceintervals and Figure 1 shows the data represented graphically for the myeloma cell line JJN3.

In vivo analysis

The results of treatment are summarized in Tables 1 and 3.

Adverse events

Transient myelosuppression was a universal feature with neutrophils<0·5#109/l following thefirst course of treatment. Mild anaemia was seen in five patients and thrombocytopenia occurredin four patients (grade 4 in two patients). Two patients were given a reduced dose of CCNU(25 mg/m2) for the second course.

Neutropenic sepsis requiring admission occurred in two patients, one requiringadmission after each course. One patient died of gastrointestinal haemorrhage with grade 4

Table 2. In vitro thymidine uptake assay

Cell line

IC50 values (ng/ml) and 95% confidence intervals (ng/ml)

U266B JJN3 U251 U937

Tumour cell type Myeloma Myeloma Glioblastoma Histiocytic lymphoma

Daunorubicin1·37

(0·492–3·811)5·597#10"2

(0·3945–7·938)5·662#10"2

(2·719–11·79)0·216

(1·232–3·789)

Doxorubicin4·19

(0·795–22·11)0·24

(0·9216–6·232)7·91#10"4

(2·299–27·22)0·127

(0·3061–5·305)

Liposomal daunorubicin80·5

(1·436–45·15)7·246

(5·695–9·22)49·65

(3·063–8·047)17·14

(0·7766–3·784)

Liposomal doxorubicin 2·85#104573·7

(3·126–10·53)1·397#104

(0·7567–2·578)5·99#103

(1·322–2·715)

Copyright ? 1998 John Wiley & Sons, Ltd. Hematol. Oncol. 16: 47–55 (1998)

52 . .

thrombocytopenia 44 days after a second course of CDD. Five patients have had follow-upechocardiograms with no change in cardiac function. Non-haematological toxicity was minimalwith nausea and fatigue seen in one patient. Alopecia was not seen.

Response rate

Four patients showed a major response with a 50 per cent reduction in marrow plasma cells.Two of these patients (both double autograft patients) subsequently relapsed 2 and 3 monthsfollowing the last course of CDD and have subsequently died of disease. The other tworesponding patients have received further treatment, one with further CDD (total of fourcourses) and the other with intermediate dose melphalan. Interestingly one of these patients hadpreviously not responded to CIDEX. Two patients showed no response to CDD and oneof these patients has subsequently died of disease. One patient died of gastrointestinalhaemorrhage prior to evaluation of response.

DISCUSSION

The in vitro data in myeloma cell lines show that daunorubicin, doxorubicin and the twoliposomal derivatives show cytotoxic activity against myeloma cell lines. There is howeversubstantially less activity shown by the pegylated doxorubicin form compared to liposomaldaunorubicin. This observation of differential cytotoxicity has recently been described for anumber of other tumour cell lines.21 There is evidence from liposomal stability studies that thedifferential cytotoxicity is due to a greater liposomal stability of the commercial doxorubicinpreparation due to its methoxypolyethylene glycol moieties. This presumably leads to a decreasein drug release and delivery in vitro. In addition native daunorubicin appears to inhibit cellproliferation to a greater extent than liposomal daunorubicin in this assay which could also beexplained by liposomal stability. These data confirm the efficacy of anthracyclines againstmyeloma cell lines in vitro and support the previous observation of a possible formulation-dependent reduction of direct in vitro cytotoxicity for pegylated liposomal doxorubicin.

The in vivo data show that the CDD regime shows efficacy in four out of seven relapsedpatients with multiple myeloma, all of whom had been heavily pretreated. One of the responding

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Figure 1. JJN3 cytotoxicity assay.

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DD regime

Patient CNeutropeniaGrade 1–4

ThrombocytopeniaGrade 1–4

AnaemiaGrade 1–4 Other

PC 100

4 2 2

JL 220

4 3 2

PT 1 4 4 2JB 3

24 1 1

LP 20

4 3 2 Reduced CCNU25 mg/m2 forsecond course

RJ 10

4 1 1

EE 00

4 4 4 Reduced CCNUto 25 mg/m2 forsecond course

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Table 3. Toxicity of C

ourses Infections Nausea Alopecia Other

7/06/978/07/975/08/97

UTI postthird course

No No

4/06/979/07/978/09/97

None No No

1/09/97 None Moderate No Fatigue0/09/971/10/97

None No No

5/11/976/01/98

Three admissionswith chestinfections

No No

2/17/977/01/98

None No No

5/01/989/02/98

None No No

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patients had previously failed to respond to four courses of CIDEX regime, supporting a definiteeffect of daunoxome. The main toxicity of the CDD regime was myelosuppression, particularlyneutropenia and two patients required admission for neutropenic fever. The myelosuppressionwas in part due to the CCNU and we reduced the dose of this from 50 mg/m2 to 25 mg/m2 in twopatients after myelosuppression was seen following the first course of treatment. Other toxicitieswere minimal and included mild nausea in one patient. Mucositis was not a feature with nograde 2–4 mucositis. Cardiac assessment was by echocardiography which showed no changebetween pretreatment and post-treatment in the five patients assessed, none of whom sufferedwith any cardiac problems. Liposomal daunorubicin given in combination with oral CCNU anddexamethasone appears to be well tolerated and efficacious in four out of the seven patients withrelapsed, advanced myeloma. Together with the in vitro data on its cytotoxicity in myeloma andits favourable pharmacokinetic profile further studies of liposomal daunorubicin in myelomawould be warranted.

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1. Dorr, R. T., Von Doff, D. D. Daunorubicin HCl. In: Dorr, R. T., Von Doff, D. D., eds. CancerChemotherapy Handbook, 2nd edn. Norwalk, CT: Appleton & Lange, 1994: 355–364..

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6. Rahman, A., White, G., More, N., Schein, P. S. Pharmacological, toxicological and therapeuticevaluation in mice of doxorubicin entrapped in cardiolipin liposomes. Cancer Res. 1985, 45, 796–803.

7. Gill, P. S., Espina, B. M., Muggia, F., et al. Phase I/II clinical and pharmacokinetic evaluation ofliposomal daunorubicin. J. Clin. Oncol. 1995, 13, 996–1003.

8. Guaglianone, P., Chan, K., DelaFlor-Weiss, E., et al. Phase I and pharmacologic study of liposomaldaunorubicin (DaunoXome). Invest. New Drugs 1994, 12, 103–110.

9. Dorr, R. T., Von Doff, D. D. Doxorubicin. In: Dorr, R. T., Von Doff, D. D., eds. CancerChemotherapy Handbook, 2nd edn. Norwalk, CT: Appleton & Lange, 1994: 638–639.

10. Forssen, E. A., Coulter, D. M., Proffitt, R. T. Selective in vivo localisation of daunorubicin smallunilamellar vesicles in solid tumours. Cancer Res. 1992, 52, 3255.

11. Forssen, E. A., Ross, M. E. Daunoxome treatment of solid tumours: preclinical and clinicalinvestigations. J. Liposome Res. 1994, 4, 481.

12. Daunoxome Reference Monograph. Nexstar Inc., 1996.13. Gill, P. S., Wernz, J., Scadden, D. T., et al. Randomised phase III trial of liposomal daunorubicin

(DaenoXome) versus doxorubicin, bleomycin, vincristine (ABV) in AIDS-related Karposi’s Sarcoma.J. Clin. Oncol. 1996, 8, 2353–2364.

14. Presant, C. A., Scolaro, M., Kennedy, P., et al. Liposomal daunorubicin treatment of HIV-associatedKarposi’s sarcoma. Lancet 1993, 341, 1242.

15. MacLennan, I. C. M., Chapman, C., Dunn, J., Kelly, K. Combined chemotherapy with ABCMversus melphalan for treatment of myelomatosis. Lancet 1992, 339, 200–205.

16. Abboudi, Z., Giles, C., Kelsey, S. M., et al. An update of oral idarubicin with CCNU anddexamethasone for relapsed myeloma: an effective oral regimen with minimal toxicity. Br. J.Haematol. 1997, 97(Suppl. 1), 86.

17. Barlogie, G., Smith, L., Alexanian, R. Effective treatment of advanced multiple myeloma refractoryto alkylating agents. N. Engl. J. Med. 1984, 310, 1353–1356.

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18. Forgeson, G., Selby, P., Lakhani, S., et al. Infused vincristine and adriamycin with high dosemethylprednisolone (VAMP) in advanced previously treated multiple myeloma patients. Br. J.Cancer 1988, 58, 469–473.

19. McBride, N. C., Richardson, D. S., Johnson, S., et al. Successful use of liposomal daunorubicin(DaunoXome) as a single agent and in combination for salvage therapy in poor prognosis lymphoma.Blood 1997, 90(Suppl. 1), 857.

20. Nikitin, E. A., Iahnina, E. I., Pivnik, A. V., Medvedev, P. V., Marjin, D. S., Astsaturov, I. A.Treatment of primary aggressive lymphoma with liposomal daunorubicin (DaunoXome) substitutionin CHOP regimen. Blood 1997, 90(Suppl. 1), 3978.

21. Wiles, M. E., Bell, C., Landfair, D., Lynam, E., Bendele, R. A. Anthracycline efficacy in vitro:cytotoxicity of liposomal/nonliposomal daunorubicin and doxorubicin for multiple tumor cell types.Drug Delivery 1997, 4, 255–262.

Copyright ? 1998 John Wiley & Sons, Ltd. Hematol. Oncol. 16: 47–55 (1998)