Br. J. Cancer (1985), 52, 841-848
`
`Poly-L-aspartic acid as a carrier for doxorubicin: a
`comparative in vivo study of free and polymer-bound drug
`G. PratesiI, G. Savi1, G. Pezzoni1, 0. Bellini2, S. Penco2, S. TinelliI &
`F. Zuninol
`
`'Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan; 2Farmitalia Carlo Erba, Nerviano, Italy
`
`Summary The synthetic polypeptide, poly-L-aspartic acid (PAA, mol. wt = 20,000) has been used as a
`macromolecular carrier for doxorubicin. The drug may be released in vivo through hydrolysis of the ester
`linkage formed between the carboxyl groups of the polymer and the drug side chain. PAA has been found to
`be a suitable carrier since it is a soluble, biodegradable, multivalent and nontoxic polymer. The toxicity and
`the therapeutic efficacy of free and polymer-linked doxorubicin have been evaluated in normal and tumour-
`bearing mice, using a variety of experimental tumour systems. In studies on single and multiple drug
`administration, the results indicated that the polymeric derivative of doxorubicin had approximately 3-fold
`lower toxicity than did free drug. In addition, the severity of specific toxic effects, including cardio- and
`vesicant toxicity, were appreciably reduced following conjugation to PAA. The doxorubicin-PAA conjugate
`gave similar or rather greater therapeutic effects than free drug at less toxic doses. This effect, more evident in
`the highly sensitive tumours, suggests an improvement of the therapeutic index of the polymer-linked drug.
`
`In an attempt to improve tumour drug uptake and,
`therefore,
`the
`selectivity of antitumour agents,
`many carrier systems have been tested. Means of
`drug
`delivery
`include
`target-specific
`biological
`carriers (Ghose et al., 1983) as well as other non-
`microparticulate,
`specific
`macromolecular
`and
`synthetic carriers (Gregoriadis, 1981; Gros et al.,
`1981). Macromolecular drug carrier systems have
`been extensively developed in an attempt to modify
`the pharmacokinetic behaviour of antitumour drugs
`(Kaye, 1981).
`Although preferential delivery of the drug to
`tumour cells remains to be documented, linkage of
`cytotoxic agents to suitable macromolecules has
`been found to improve therapeutic efficacy (Arnon
`& Hurwitz, 1983). In some cases, the therapeutic
`advantage
`of the macromolecular derivative
`is
`related to reduction of systemic drug toxicity, thus
`allowing the administration of higher doses (Levi-
`Schaffer et al., 1982).
`Recently, we have reported that daunorubicin
`covalently linked to poly-L-aspartic acid (PAA)
`reduce the toxicity of the anthracycline, whereas it
`maintained or improved the antitumour efficacy
`(Zunino et al., 1982, 1984). Doxorubicin may be
`released in
`vivo through hydrolysis of the ester
`linkage formed between the carboxyl group of the
`polymer and drug side chain. These results have
`generated considerable interest, since the polymeric
`drug form might be of potential clinical relevance.
`
`Correspondence:
`F. Zunino
`Received 15 April 1985; and in revised form, 19 August
`1985.
`
`Thus, the present studies were initiated to further
`document the preclinical efficacy of doxorubicin
`conjugated with PAA. Polymer-bound doxorubicin
`was compared to free drug with respect to some
`toxic
`effects.
`addition,
`In
`this paper describes
`detailed in vivo evaluation of free and PAA-bound
`doxorubicin against experimental tumour systems
`with particular reference to solid tumours.
`
`Materials and methods
`
`Drugs
`Daunorubicin, doxorubicin and 14-bromo-dauno-
`rubicin were supplied by Farmitalia Carlo Erba
`(Milan, Italy) as hydrochlorides. Drug solutions
`were freshly prepared immediately before use. PAA
`(mol.
`20,000)
`obtained
`Sigma
`from
`wt
`was
`Chemical Co. (St Louis, Mo., USA). Doxorubicin-
`PAA conjugate (poly-L-aspartic acid doxorubicinyl
`ester; previously referred to as daunorubicin-PAA
`conjugate since a daunorubicin derivative was used
`in
`the
`conjugation
`procedure)
`was
`prepared
`essentially according to the previously described
`procedure (Zunino et al., 1982, 1984). Attachment
`of drug to PAA was achieved by nucleophilic
`substitution
`reaction
`of 14-bromo-daunorubicin.
`Thus, an ester linkage was formed between the
`drug side
`chain and carboxyl groups of the
`polyamino acid. The various preparations of doxo-
`rubicin-PAA conjugate used in this study contained
`18-70 mol drug mol-1 of PAA.
`The
`concentration
`of
`anthracycline
`in
`the
`polymeric derivative was determined by absorbance
`
`©) The Macmillan Press Ltd., 1985
`
`SANOFI-AVENTIS Exhibit 1042 - Page 841
`
`IPR for Patent No. 8,951,962
`
`

`
`842
`
`G. PRATESI et al.
`
`at 495 nm. The conjugate was stable in aqueous
`solution for at least 1 month as checked by thin-
`layer chromatography, using a mixture of chloro-
`form:methanol:acetic acid (80:20:4) as a solvent.
`In all experiments, the dose of the do:;orubicin-
`PAA conjugate was expressed as drug content in
`the polymeric derivative.
`
`Animals
`sexes employed
`of both
`The mice and rats
`throughout the experiments were obtained from
`Charles River Laboratories (Calco, Como, Italy).
`Mice weighed between 17 and 22g.
`
`Evaluation of toxicity
`Drug-induced mortality was assessed in healthy
`C3H/He mice treated i.v. with single or multiple
`doses of doxorubicin, and doxorubicin-PAA and
`followed for 110 days, since mice that did not die
`approximately
`after
`died
`2-3
`soon
`treatment
`months later. At necropsy, mice showed spleen and
`liver size reduction and haemorrhagic degeneration
`of intestinal mucosa.
`In tumour-bearing mice, deaths that occurred in
`before
`the
`first
`death
`of an
`treated
`animals
`untreated control were attributed to drug toxicity.
`Normal healthy mice were treated i.v. according to
`the same schedules used for the solid-tumour-
`bearing mice, and recorded for 120 days.
`Cardiotoxicity was assessed in healthy female
`according
`the same
`treated
`C3H/He mice
`to
`for
`used
`of
`schedule
`mammary-
`treatment
`carcinoma-bearing mice (q7d x 4 i.v.). Forty and 90
`days after the last treatment, mice were killed and
`hearts were removed and fixed in paraformaldehyde
`(4% in 0.1 M phosphate buffer at pH 7.3). Histo-
`logical examination of semi-thin sections was carried
`out as previously described (Bertazzoli et al., 1979).
`were graded
`according
`lesions
`Myocardial
`to:
`degree). The histo-
`(severity degree) x (extension
`logical examinations were blind.
`The vesicant activity was assessed in Sprague-
`Dawley rats weighing about 250 mg. Rats were
`injected i.d. in both flanks with 1 ml of a solution
`containing different drug concentrations in distilled
`water. The healing scar was measured on day 12 on
`its largest dimension and was scored as follows:
`2+ =6-lOmm;
`I + = _Smm;
`3+ = >lOmm.
`Scores were added and expressed as a response
`fraction (RF) of the possible total cumulation score
`of 3 + for each rat (Jenkins & Corden, 1983).
`
`Tumours
`The macrophage tumour J774 was serially main-
`tained in ascitic form in female BALB/c mice. For
`chemotherapy experiments,
`106
`cell/mouse were
`
`BALB/c
`female
`in
`i.p.
`injected
`or CDF1
`(BALB/c x DBA/2) mice (Tarnowski et al., 1979).
`Lewis lung carcinoma was serially maintained
`according to Geran et al. (1972). The experiments
`were carried out in BDF1 (C57BL x DBA/2) mice,
`inoculated i.m. with 5 x 105 cells/mouse.
`M5076/73A (M5),
`reticulum
`cell
`murine
`a
`sarcoma, was transplanted i.m. in the right hind leg
`of female C57BL mice by injection of 5 x 105
`cells/mouse, for serial passages and chemotherapy
`experiments (Talmadge et al., 1981). In the ascitic
`form, 2 x 106 cells/mouse were implanted i.p. in
`female BDF1 mice.
`Mammary adenocarcinoma, spontaneously arisen
`female
`C3H/He
`a
`retired
`in
`breeder,
`was
`transplanted in the left axillary region in female
`C3H/He mice. The chemotherapy experiment was
`carried out on second generation transplant in
`C3H/He
`injected
`female
`mice
`with
`2 x 107
`cells/mouse (Di Marco et al., 1972).
`
`Evaluation of antitumour activity
`Unless otherwise indicated, for antitumour activity
`experiments 10mice/group were used. The effect on
`survival is expressed as percentage ILS (increase
`calculated
`in
`life
`span)
`as
`follows:
`ILS=
`[(T/C) -1] x 100, where T/C is the median survival
`time (MST) of dying mice only in the treated
`group (T) divided by the MST of the untreated
`control group (C). Long-term (at least 90 days)
`survivors (LTS) were considered cured and were
`noted separately.
`In the mice injected with solid growing tumours,
`tumour growth was assessed by weekly caliper
`measurement of the two tumour diameters and
`tumour weight was obtained according to Geran et
`al. (1972). In experiments carried out against early
`tumour, the anti-tumour activity was established by
`the percentage of tumour growth inhibition of the
`treated mice as compared to the controls at the day
`indicated in each experiment.
`In the experiments designed to evaluate anti-
`tumour activity against advanced tumours, tumour
`weight in individual mice was determined at the
`beginning of treatment, and tumour growth was
`then evaluated for individual mice as the percentage
`change in tumour weight 1 week after the last
`treatment. The data reported as relative tumour
`weight represent the average of individual tumour
`weight change for each group.
`Student's
`test
`t
`was
`comparisons.
`
`used
`
`for
`
`statistical
`
`Results
`
`Toxicity in non-tumour-bearing mice
`Table I shows that linkage of the anthracycline to
`
`SANOFI-AVENTIS Exhibit 1042 - Page 842
`
`IPR for Patent No. 8,951,962
`
`

`
`PAA AS A DOXORUBICIN CARRIER
`
`843
`
`Table I
`
`Lethal toxicity of free and poly-L-aspartic acid (PAA)-
`bound doxorubicin (DX)
`
`Mouse
`strain
`
`Drug
`
`Dosea
`
`No. of
`treatments
`
`Deaths
`
`Survival
`range (d)
`
`C3H/He
`males
`
`DX
`
`DX-PAA
`
`DX
`
`DX-PAA
`
`C3H/He
`females
`
`C57BL
`females
`
`DX
`
`DX-PAA
`
`13
`1
`0/8
`16.9
`1
`4/8
`8/8
`22
`1
`22
`0/8
`1
`0/8
`28.5
`1
`2/8
`37
`1
`6
`4
`3/15
`7.5
`4
`9/14
`15
`4
`0/5
`18
`4
`4/15
`21.5
`10/10
`4
`6
`3/10
`3
`7.5
`9/10
`3
`10/10
`9
`3
`14.4
`3
`0/10
`18
`3
`0/10
`22.5
`3
`1/10
`22
`amgkg-linjection-1, i.v. In the case of the DX-PAA conjugate,
`the dose refers to the actual amount of drug in the conjugate.
`
`16-52
`5-68
`
`22-45
`88-90
`15-90
`
`30-87
`22-105
`5-11
`5-65
`11-81
`
`PAA markedly reduced drug toxicity in different
`mice strains after i.v. administration of single or
`multiple weekly doses. After a single i.v. injection
`of the drugs to male C3H/He mice, the LD2 s
`values were -15 and 37mgkg-' for doxorubicin
`and doxorubicin-PAA, respectively. Thus, the ratio
`between these equitoxic doses of doxorubicin-PAA
`and free doxorubicin was 2.5.
`The chronic lethal toxicity of the drug on the
`same strain caused by 4 weekly i.v. injections could
`be further reduced when given in the polymeric
`form; thus, the ratio between equitoxic doses of
`doxorubicin-PAA (18mgkg-1) and doxorubicin
`(6mg kg- 1) was -3 in this experiment. Moreover,
`in C57BL mice, doxorubicin-PAA conjugate seemed
`definitely better tolerated than free doxorubicin
`using a multiple treatment schedule (q7d x 3, i.v.); in
`this strain, the ratio between equitoxic doses was
`more than 3 (doxorubicin-PAA 22.5mg kg' vs.
`doxorubicin < 6 mg kg - 1).
`Data from the cardiotoxic test in C3H female
`mice (Table II) showed that the linkage of the
`anthracycline to PAA also reduced this organ-
`specific damage. The dose of doxorubicin-PAA
`be
`increased
`3-fold
`relative
`must
`free
`to
`doxorubicin in order to produce a comparable
`effect in the heart. This parallels the effect on
`mortality.
`The ulcerogenic potential of doxorubicin and its
`polymeric derivative was assessed
`in Sprague-
`Dawley rats (Table III). The vesicant action of
`
`Table II
`
`Cardiotoxicity in C3H/He mice
`Lesion gradec at dayd
`40e
`
`90
`
`Druga
`
`Doseb
`
`LA'
`
`Vg
`
`LA
`
`V
`
`0.7
`1.7
`0.3
`0.4
`
`1.5
`5.0
`0.6
`1.2
`
`DX
`
`DX-PAA
`
`6
`7.5
`15
`18
`21.5
`
`1.2
`2.3
`0.7
`0.6
`1.3
`
`1.9
`3.3
`0.8
`1.6
`3.2
`aDX,
`poly-L-aspartic
`doxorubicin;
`PAA,
`acid;
`bmg kg- 'injection-', q7q x 4 i.v. In the conjugate, the
`dose is expressed as dose of drug component; cGiven by
`product
`the
`(severity
`of
`degree) x (extension
`degree);
`dCalculated from the last treatment; eData collected from
`two experiments (5 mice/group). One experiment was
`carried out in parallel with anti-tumour activity assay
`(Table VIII); 'Left atrium; gVentricles.
`
`2.4mg of doxorubicin-PAA was lower than that
`induced by 0.6mg of doxorubicin. The doxorubicin-
`PAA conjugate at lower dose (1 mg) did not show
`appreciable vesicant activity.
`
`Anti-tumoural activity studies
`Since in the treatment of drug-sensitive tumours,
`the antitumour effects of the doxorubicin-PAA
`
`SANOFI-AVENTIS Exhibit 1042 - Page 843
`
`IPR for Patent No. 8,951,962
`
`

`
`844
`
`G. PRATESI et al.
`
`Table III
`Vesicant
`activity
`of
`doxorubicin
`(DX) and the DX-poly-L-aspartic acid (PAA)
`conjugate
`
`Drug
`
`DX
`
`DX-PAA
`
`Amount
`(mg)
`
`No. of
`rats
`
`1
`0.6
`2.4
`1
`
`6
`8
`6
`2
`
`RFa
`
`0.83
`0.70
`0.50
`0.00
`
`aResponse
`fraction.
`methods for details.
`
`See
`
`Materials
`
`and
`
`conjugate
`found
`dose-dependent
`be
`to
`were
`(Zunino et al., 1982, 1984) and, as already observed
`for other antitumour drugs, optimal treatment was
`at the maximum tolerated doses, in antitumour
`activity experiments the dose levels were usually
`selected in the range of the highest non-toxic doses
`(.LD1O). The relative effectiveness of doxorubicin,
`
`in the survival time than did doxorubicin. However,
`the difference was not statistically significant.
`Table VI shows the effect of doxorubicin and
`doxorubicin-PAA on i.m. implanted M5 tumour in
`female C57BL mice using a multiple treatment
`schedule (q7d x 3,
`i.v.)
`starting on day 1
`after
`tumour implant. Doxorubicin-PAA gave a complete
`inhibition of tumour growth at the LD1O (i.e.,
`22.5mgkg-1) and even at a lower dose. However,
`doxorubicin produced complete inhibition only at
`(7.5
`toxic
`doses
`and 9mgkg- 1).
`Both
`the
`compounds slightly increased the survival time of
`tumour-bearing mice.
`The effects of i.v. treatments (q3d x 4, beginning
`on day 1 after tumour implant) with doxorubicin
`and polymeric derivative on Lewis lung carcinoma
`are shown in Table VII. Doxorubicin was active in
`inhibiting tumour growth at 5 mg kg- 1. Its activity
`was not statistically different from that produced
`by doxorubicin-PAA at 18mg kg-1. By 120 days, 9
`of 10 of the doxorubicin-(7.5 mg kg- 1)-treated mice
`survived, whereas in another experiment 7 of 10
`
`Table IV
`
`Anti-tumoural activity against i.p. J774 tumour
`
`Toxic
`deathsd
`
`LTSC
`
`Drugsa
`
`DX
`
`DR
`
`DX-PAA
`
`Doseb
`
`6.6
`10
`3.3
`5
`30-32
`40-45
`
`ILS
`(Y)c
`123 (68,178)
`1/18
`3/18
`94 (76,112)
`0/18
`5/18
`39 (39,40)
`1/18
`0/18
`-15 (-29,0)
`8/18
`0/18
`102 (113,91)
`2/18
`2/18
`-21 (-40, -2)
`10/18
`3/18
`aDX, doxorubicin; DR, daunorubicin; DX-PAA, doxorubicin-poly-
`L-aspartic acid conjugate; bmgkg-1, treatment i.p. on day 1. Dose
`refers to actual amount of drug in the conjugate; cln parenthesis, the
`values of each experiment. MST of the control mice was 19 and 23.5
`days in the two experiments. Nine mice/group were used in each
`experiment; dEvaluated in tumour-bearing mice; eEvaluated 90 days
`after inoculation of tumour cells.
`
`daunorubicin
`doxorubicin-PAA
`and
`in
`the
`treatment of early ascitic J774 tumour is presented
`in Table IV. When administered as a single dose
`i.p. one day after cell inoculation, daunorubicin
`slightly increased the survival time at the optimal
`dose of 3.3 mg kg- 1. Doxorubicin and doxorubicin-
`PAA, at their respective optimal doses (6.6 and
`32mgkg-1), markedly increased the survival time
`of the tumour-bearing mice, producing also long-
`survivors.
`Although
`the
`activity
`term
`was
`comparable for the two drugs, doxorubicin-PAA
`gave more reproducible effects.
`After a single i.p. treatment of M5 ascitic tumour
`(Table V), doxorubicin-PAA gave a higher increase
`
`Table V Anti-tumour activity against M5 ascitic
`tumour
`
`DX
`
`DX-PAA
`
`Druge
`
`Doseb
`
`Toxic
`deaths'
`
`ILS
`(Y)C
`6.6
`63
`1/10
`10.0
`54
`1/10
`26.0
`0/10
`90
`32.0
`107
`0/10
`aDX, doxorubicin; DX-PAA, doxorubicin-poly-L-
`aspartic acid conjugate; bmg kg-', treatment i.p. on
`day 1. Dose refers to actual amount of drug in the
`conjugate; CMST of the control mice was 22 days;
`dEvaluated in tumour-bearing mice.
`
`SANOFI-AVENTIS Exhibit 1042 - Page 844
`
`IPR for Patent No. 8,951,962
`
`

`
`PAA AS A DOXORUBICIN CARRIER
`
`845
`
`Table VI
`
`Anti-tumour activity against M5 solid tumour
`
`7iTmour weightc
`
`ILS (%)d
`
`Druga
`
`Doseb
`
`Exp. I
`
`Exp. 2
`
`Exp. I
`
`Exp. 2
`
`Toxice
`deaths
`
`DX
`
`DX-PAA
`
`6
`7.5
`9
`11.2
`
`270+400 (92)
`
`0
`
`(100)
`
`140+200 (95)
`(100)
`0
`
`1520+300 (42)
`790+360 (70)
`
`3/10
`9/10
`10/10
`0/10
`0
`17.2
`(100)
`0/10
`18
`22.5
`0
`(100)
`39
`1/10
`aDX, doxorubicin; DX-PAA, doxorubicin-poly-L-aspartic acid conjugate; bmgkg- injection-1,
`q7d x 3, i.v., starting from day 1. In the conjugate, dose refers to actual amount of drug; cMean± s.d.
`measured on day 21 after tumour transplantation; in parenthesis, percentage inhibition. The average
`tumour weight of control mice was 2610( ± 500) mg (experiment 1) and 3621 (± 770) mg (experiment
`2); dMST of control mice was 32.5 (experiment 1) and 31 days (experiment 2). Ten mice per group
`were used; 'Evaluated at 120 days in healthy female C57BL mice.
`
`15
`
`32
`
`21
`38
`
`17
`
`20
`
`Table VII
`
`Anti-tumour activity against Lewis lung carcinoma
`
`Drug'
`
`Doseb
`
`Tumour
`weight (mg)c
`
`Tumour growth
`inhibition (%)
`
`ILS
`(%)d
`
`Toxic
`deathse
`
`DX
`
`DX-PAA
`
`5
`7.5
`12
`18
`
`730+710
`0
`2660+990
`380+410
`
`87
`60
`1/10
`ND'
`100
`ND
`53
`20
`0/10
`93
`0/10
`86
`'DX, doxorubicin, DX-PAA, doxorubicin-poly-L-aspartic acid conjugate;
`bmgkg- injection-1, q3dx4,i.v. starting from day 1. In the conjugate, the
`dose is expressed as actual amount of drug; cMean+s.d. measured on day 21
`after tumour transplantation. The average tumour weight of control mice was
`5647(±2710)mg; dMST of control mice was 22.5 days; eIn tumour-bearing
`mice; 'Not determined. See Results for details.
`
`mice died from toxicity. Both the compounds had a
`similar effect on survival time.
`Table VIII compares the activity of doxorubicin
`and doxorubicin-PAA in the treatment of advanced
`(-.75 mg)
`mammary carcinoma
`implanted
`in
`C3H/He mice, with drug administration on the
`q7d x 4
`(i.v.)
`schedule.
`At
`equitoxic
`doses
`(doxorubicin, 6mg kg-1
`doxorubicin-PAA,
`and
`18 mg kg 1), the conjugate produced a somewhat
`greater
`inhibition
`tumour
`(82%)
`than
`that
`produced by free doxorubicin (63%), although the
`difference
`was
`statistically
`not
`significant.
`Moreover, doxorubicin-PAA produced a similar
`inhibition (76%) at a non-toxic dose (15mgkg-1).
`Both drugs were ineffective in increasing survival
`time.
`
`Discussion
`
`The results presented in this study indicate that, in
`comparison to free drug, administration of doxo-
`
`rubicin covalently linked to the anionic polyamino
`acid, PAA, resulted in reduced toxicity, after single
`and
`after
`multiple
`administrations.
`Decreased
`systemic toxicity paralleled reduction in severity of
`specific
`effects,
`toxic
`including
`cardiotoxicity,
`probably related to decreased heart uptake of the
`drug (Mazzoni et al., in preparation). This obser-
`vation is of particular interest, since potentially
`irreversible cardiac damage is
`the major dose-
`limiting toxicity for doxorubicin (Lenaz & Page,
`1976). In particular, since in the same experiment
`(using healthy and tumour-bearing CH3 mice) we
`could compare cardiotoxic, lethal and anti-tumour
`effects, a therapeutic improvement following linkage
`of the anthracycline to
`the polymer could be
`directly shown (Tables
`II and VIII).
`Indeed,
`although
`equitoxic
`doses
`of free
`doxorubicin
`(6mg kg- 1) and doxorubicin-PAA (18mg kg- ')
`caused comparable heart lesions and had similar
`effects on tumour growth, the doxorubicin-PAA
`conjugate retained anti-tumour activity at a lower
`
`SANOFI-AVENTIS Exhibit 1042 - Page 845
`
`IPR for Patent No. 8,951,962
`
`

`
`846
`
`G. PRATESI et al.
`
`Table VIII
`
`Anti-tumour activity against advanced C3H mammary carcinoma
`
`Drug'
`
`Doseb
`
`Relative
`tumour weight
`(%) C
`
`Tumour growth
`inhibition
`(O)
`
`ILS
`(%)d
`
`Toxic
`deathse
`
`DX
`
`DX-PAA
`
`6
`7.5
`15
`18
`21.5
`
`63
`2751+1640
`91
`668+ 490
`1810+ 905
`76
`82
`1326+ 865
`ND'
`ND
`DX-PAA, doxorubicin-poly-L-aspartic
`doxorubicin;
`conjugate;
`"DX,
`acid
`bmg kg- 1 injection'- , q7d x 4, i.v., starting when tumour weight was - 75 mg. In the
`conjugate, the dose is expressed as dose of drug component; CMean + s.d. measured
`on day 48 after tumour transplantation (1 week after the last treatment); the relative
`tumour weight for control mice was 7411 (± 3092)%; dMST of control mice was 74
`days. Nine mice per group were used; eEvaluated at 120 days in healthy female
`C3H/He mice; fND, not determined.
`
`-7
`12
`-6
`8
`ND
`
`3/15
`9/14
`0/5
`4/15
`10/10
`
`was well
`cardiotoxic
`
`(15mgkg-1).
`dose
`This
`dose
`level
`tolerated and definitely caused less
`damage.
`In addition, the marked attenuation of dermal
`toxicity of the drug in the polymeric derivative
`might
`have
`obvious
`implications.
`practical
`Reduction of local toxicity is also consistent with
`the observation that high doses of the conjugate
`well
`tolerated
`after
`i.p.
`administration
`were
`(Zunino,
`1982). Again, this might have clinical
`relevance because of the therapeutic potential of
`doxorubicin in the intraperitoneal chemotherapy of
`abdominal neoplastic diseases (Myers & Collins,
`1983).
`The anti-tumour activity, evaluated in a number
`of experimental models, indicated that doxorubicin
`linked to the polyamino acid provided therapeutic
`effects similar to those of free drug at less toxic
`doses. In fact, as summarised in Table IX, the data
`obtained in the treatment of three solid growing
`
`tumours do show a trend toward an improved
`therapeutic index of the drug following polymer
`linkage, although a quantitative assessment of this
`improvement is difficult to make due to the limited
`number of doses used in these experiments. An
`improvement in
`the
`therapeutic index resulting
`from conjugation was more evident in the MS
`model. Further studies aimed at other tumour
`models and different schedules would therefore be
`appropriate. Thus, although the covalent linkage of
`the drug to polymer also resulted in a reduction in
`drug potency, the therapeutic advantage, reflected
`by a greater margin of safety, was generally
`observed in a variety of experimental models
`(Zunino et al., 1982, 1984).
`Moreover,
`the
`indicated
`results
`that
`the
`doxorubicin-PAA conjugate was also superior to
`free drug against MS tumour, a reticulum cell
`(Talmadge
`al.,
`1981).
`remains
`It
`et
`sarcoma
`uncertain whether the increased therapeutic efficacy
`
`Table IX Comparison of therapeutic ratios of free and PAA-bound
`doxorubicin
`
`Effective
`LDIO
`dose
`(mgkg- )b (mgkg-')
`
`Therapeutic
`ratioc
`
`Druge
`DX
`DX-PAA
`DX
`DX-PAA
`DX
`DX-PAA
`
`Experimental model
`(solid tumours)
`
`M5
`
`Lewis lung
`
`Advanced C3H
`Mammary carcinoma
`
`<1
`6
`< 6
`17.2
`22.5
`1.3
`5
`5
`1
`>1
`18
`> 18
`7.5
`< 6
`<0.8
`18
`> 15
`>0.8
`doxorubicin-poly-L-aspartic
`aDX,
`doxorubicin;
`DX-PAA,
`acid
`conjugate; bdose providing a tumour growth inhibition _ 80% as
`compared to untreated mice; CRatio between LDIo and effective dose.
`
`SANOFI-AVENTIS Exhibit 1042 - Page 846
`
`IPR for Patent No. 8,951,962
`
`

`
`to PAA, in
`linked
`the
`of
`this
`anthracycline
`model,
`is due to
`its
`phagocytic
`experimental
`properties (Talmadge et al., 1982), since apparently
`free and polymer-linked drug displayed comparable
`against
`activity
`the macrophage tumour J774.
`However, it should be noted that in two separate
`experiments (Table IV), the efficacy of free doxo-
`rubicin in increasing survival time was markedly
`different, in contrast to the reproducible effect of the
`polymeric derivative. Thus, no definitive conclusions
`could be drawn from these experiments. Relevant to
`this point is the observation that in a variety of
`experimental
`models,
`reproducible
`results
`were
`generally obtained using different preparations of
`the conjugate. This also reflects the stability of the
`conjugate in aqueous solutions.
`Taken together with previous results showing an
`enhanced efficacy of the polymeric derivative as
`compared to free doxorubicin against leukaemia
`models (Zunino et al., 1984), the data presented
`here suggest a therapeutic advantage of the drug
`linked
`to PAA in
`tumours
`of
`tissues
`of
`mesenchymal origin. Anyway, the attachment of
`doxorubicin to this macromolecular carrier did not
`cause loss or reduction of anti-tumour efficacy at
`optimal doses in any of the tumour models used.
`However, this means of drug delivery
`is not
`expected to overcome drug resistance, since free
`and
`polymer-linked
`doxorubicin
`produced
`a
`marginal response against colon carcinoma 26 (not
`shown), a tumour model, which, when growing
`subcutaneously, is
`relatively resistant to anthra-
`
`References
`
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`(1983). Antibody- and
`polymer-drug conjugates. In Targeted Drugs, Goldberg
`(ed) p. 23. John Wiley & Sons: New York.
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`4'-
`deoxydoxorubicin in comparison with doxorubicin.
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`DI MARCO, A.,
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`PAA AS A DOXORUBICIN CARRIER
`
`847
`
`cycline therapy (Casazza et al., 1983). This is in
`agreement with the absence of appreciable activity
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`leukaemia
`(Gross
`of highly
`sensitive
`tumours
`[Zunino et al., 1984] and M5 reticulum cell sarcoma
`[Table VI]).
`Further studies on the preclinical toxicity of this
`polymeric derivative are needed to fully evaluate
`potential of this drug delivery
`therapeutic
`the
`system.
`
`This work was supported in part by a research grant from
`the Consiglio Nazionale delle Ricerche, Rome (Progetto
`Finalizzato Oncologia). We thank Ms B. Johnston for
`editorial assistance and manuscript preparation.
`
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`SANOFI-AVENTIS Exhibit 1042 - Page 848
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`IPR for Patent No. 8,951,962