WO2003043584A2 - Engineered liposomal particles containing core-loaded pro-drugs for the controlled release of camptothecins - Google Patents

Abstract

A liposome composition includes an entrapped camptothecin ester derivative loaded predominantly in the aqueous core of the liposomal particle. More particularly the invention relates to controlled release formulations that entail the liposomal core-loading of camptothecin-20-esters (CPT-20-OR). Through judicious choice of liposomal encapsulation methodologies, lipid ingredients, and the choice of the R=CO[CH2]nNH2HC1 functionally of the CPT-20-OR, formulations are developed which release camptothecin pro-drug from the liposomal particle. Upon reaching the outside of the particle and gaining exposure to the physiological mileau (i.e. pH 6.8 to 7.4) at the tumor site, the glycinate esters decompose to generate the active lactone of camptothecins which are potent inhibitors of DNA topoisomerase I and potent anti-cancer agents.

Description

ENGINEERED LIPOSOMAL PARTICLES CONTAINING CORE-LOADED PRO-DRUGS FOR THE CONTROLLED

RELEASE OF CAMPTOTHECINS

This application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/331,908 filed November 20, 2001.

Technical Field

The present invention relates to camptothecin pro-drugs comprising camptothecin-20-aminoester derivatives loaded into the aqueous core of a liposome. The invention further relates to methods for reducing toxicity and extending in vivo survival of a camptothecin or camptothecin analogue, comprising synthesizing camptothecin-20-aminoester derivatives and loading them into the aqueous core of a liposome. Still further, the invention relates to pharmaceutical compositions comprising the camptothecin pro-drugs as described, and to methods of inhibiting topoisomerase I and treating cancer in a mammal using the camptothecin pro-drugs.

Background of the Invention

Camptothecin and related analogs (Figure 1) represent an important

class of agents useful in the treatment of cancer. The widespread clinical

interest in the camptothecins stems from their unique mechanism of action:

they stabilize the covalent binding of the enzyme topoisomerase I (topo I), an

intranuclear enzyme that is overexpressed in a variety of tumor lines, to DNA. This drug/enzyme/DNA complex leads to reversible, single strand nicks that,

according to the fork collision model, are converted to irreversible and lethal

double strand DNA breaks during replication. Therefore, due to the

mechanism of its cytotoxicity, CPT is S-phase specific, indicating that it is

only toxic to cells that are undergoing DNA synthesis. Rapidly dividing

cells, such as cancerous cells, spend more time in the S-phase relative to

healthy tissues. Thus, the overexpression of topo I, combined with the

faster rate of mitosis, provide a limited basis for selectivity via which

camptothecins can effect cytotoxicity on cancerous cells rather than healthy

host tissues.

As a class, the camptothecins have exhibited unique dynamics and

reactivity in vivo, both with respect to drug hydrolysis and blood protein

interactions. These factors have confounded their pharmaceutical development

and clinical implementation. In terms of hydrolysis, each of the clinically

relevant camptothecins shown in Figure 1 contains an α-hydroxy-δ-lactone

pharmacophore; at pH 7 and above this functionality is highly reactive and

readily converts to the "ring opened" carboxylate form (as is shown for

camptothecin in Figure 2). Unfortunately, the carboxylate form of the

camptothecin agent is inactive. Thus, as a result of the labile α-hydroxy-δ-

lactone pharmacophore, camptothecins exist in an equilibrium consisting of

two distinct drug species: 1) the biologically active lactone form where the lactone ring remains closed; and 2) a biologically-inactive carboxylate form

generated upon the hydrolysis of the lactone ring of the parent drug.

This hydrolysis problem with camptothecin and many analogs (e.g.

9-aminocamptothecin, 9-nitrocamptothecin) is exacerbated in human blood.

In human blood and tissues, the camptothecin equilibrium of active lactone

form vs. inactive carboxylate form can be greatly affected by the presence of

human serum albumin (HSA). Time-resolved fluorescence spectroscopic

measurements taken on the intensely fluorescent camptothecin lactone and

camptothecin carboxylate species have yielded direct information on the

differential nature of these interactions with HSA. The lactone form of

camptothecin binds to HSA with moderate affinity yet the carboxylate form of

camptothecin binds tightly to HSA, displaying a 150-fold enhancement in its

affinity for this highly abundant serum protein. Thus, when the lactone form

of camptothecin is added to a solution containing HSA, the preferential

binding of the carboxylate form to HSA drives the chemical equilibrium to the

right, resulting in the lactone ring hydrolyzing more rapidly and completely

than when camptothecin is in an aqueous solution without HSA.

These dynamic processes present a major hurdle to achieving

successful chemotherapy for a cancerous disease state. I n a d d i t i o n t o

modulating human blood stability, it has been shown that the presence of

physiologically relevant concentrations of HSA can greatly attenuate (by several orders of magnitude) the anticancer activities (IC₅₀ values) of these

agents. In humans it appears that protein binding interactions make it

difficult to achieve therapeutically effective unbound lactone levels of these

agents, particularly when one considers that continuous exposures (for tumor

cells to cycle through S-phase) of the active lactone form are requisite for

efficacy purposes.

Liposomes have been shown to provide an excellent means by which to

stabilize the biologically-active lactone form of camptothecins. Water-soluble

drugs such as topotecan can be entrapped within the pH-adjusted aqueous

compartments of liposomes with the acidic microclimate of liposomes

stabilizing the active lactone form. Lipophilic drugs can partition into the

bilayer where the lactone ring is stabilized and protected from hydrolysis.

Liposomes can also serve as controlled release depots. The camptothecins are

S-phase specific drugs, and it has been shown that optimal activity is obtained

when the tumors of a patient are exposed to the drugs for continuous periods

of time. Liposomes that target tumors and slowly release drug (such that tumor

cells are continuously exposed to drug) appear as attractive drug delivery

systems to pursue.

The first FDA approval of a liposomal anticancer product was

liposome-encapsulated doxorubicin (Doxil) from SEQUUS (now ALZA). In

early 1996 liposome-encapsulated daunorubicin (DaunoXome) from Nexstar (now Gilead) was also approved for marketing. Both products use

unilamellar liposome configurations composed of phosphatidylcholines

with saturated fatty acid chains and cholesterol as their basic ingredients.

These lipids in combination create a durable bilayer that prevents the leakage

of drug from the vesicle. The tough bilayer also deters the opsonization

process from occurring. The Doxil formulation includes 5%

polyethyleneglycol (PEG)-linked distearoyl-phosphatidyethanolamine in the

liposome, which effectively extends the lifetime of the liposome in

circulation. Studies have shown that the Doxil and Daunoxome products pass

through the fenestrated vasculature of growing tumors; thus, the leaky nature

of the vasculature promotes drug accumulation. An excellent example of this

is Kaposi's sarcoma, a common cancer in patients with AIDS, that is known to

respond well to tumor-targeted liposomes; the enhanced accumulation of drug

at the tumor site has been directly attributed to the leaky blood vessels that are

characteristic of Kaposi's sarcoma. The successful use of the Doxil and

Daunoxome medications described above (and other favorable clinical results

obtained with TLC-99 liposomal doxorubicin) has been achieved as a result of

30 years of intensive and persistent research and development.

Clearly, the past several years have seen rapid advancement and

progress in both the camptothecin drug development field as well as the

liposomal drug delivery arena. A number of companies have been active in the liposomal formulations of camptothecins in recent years. The lead

liposomal camptothecin is GG 211. Both Gilead and ALZA developed

liposomal formulations of this agent. Both companies reported encouraging

findings that liposomal formulation resulted in 3 to 5-fold gains in the

efficacy of the agents against human cancers carried in murine models. In

addition to GG211, Inex has reported favorable results in formulating

topotecan in liposomes, showing a significant improvement in efficacy

through the liposomal formulation process. There is also significant interest in

liposomal aerosol formulations of 9-AC and 9-NC, although these drugs only

load into the bilayer compartment of liposomes since they lack a basic amino

functionality necessary for core-loading. Core-loaded liposomal formulations

of CPT-11 have also been prepared and evaluated. Liposomal core-loading of

camptothecins has been limited to date to agents such as topotecan, CPT-11_%

GG-211, and CKD602 which actively load using ion gradients into pre-made

liposomes.

Summary of the Invention

In accordance with the purposes of the present invention as described

herein, the present invention provides a composition comprising a

camptothecin-20-aminoester derivative having the structure:

wherein R¹ may be hydrogen, a halogen atom, a branched or linear

alkyl group, a branched or linear alkenyl group, a C₃.₇ cycloalkyl group, a

branched or linear alkynyl group, an alkoxy group, an alkylamino group, a

dialkylamino group, an alkylthiol group, a thiol group, a phenyl group, an

amino group, a nitro group, a cyano group, or (CH₂)γNR₈R₉, wherein: (a) Y is

an integer from 1-10 and R_g and R₉ are, independently, hydrogen, an alkyl

group, an alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl

amine, a hydroxy group, an alkoxy group, an acyl group, or a carbamate, and

(b) R₈, Rg and the nitrogen to which they are attached may form a saturated or

unsaturated three- to ten-membered heterocyclic ring containing O, S, and

NR¹⁰ wherein R¹⁰ is a hydrogen, an alkyl group, an alkenyl group, an alkynyl

group, an alkoxy group or a carbamate. R¹ may also be a C_M0 cycloalkyl

group, a C,.₁₀ cycloalkenyl group, or a C,.₁₀ cycloalkynyl group. R² may be hydrogen, a halogen atom, a linear or branched alkyl group,

a linear or branched alkenyl group, a linear or branched alkynyl group, an

amino group, an alkylamino group, a dialkylamino group, a nitro group, a 3-10

membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl

group, a C₃.₁₀ cycloalkynyl group, a thiol group, or a cyano group. R² may

also be (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-10; and (b) R₈ and

R₉ are, independently, hydrogen, an alkyl group, an alkenyl group , an alkynyl

group, an amine, an alkyl amine, a dialkyl amine, a hydroxy group, an alkoxy

group, an acyl group, or a carbamate; and (c) R₈, Re, and the nitrogen to which

they are attached may form a saturated or unsaturated tliree to ten membered

heterocyclic ring.

R³ may be hydrogen, a halogen atom, a linear or branched alkyl group,

a linear or branched alkenyl group, a linear or branched alkynyl group, an

amino group, an alkylamino group, a dialkylamino group, a nitro group, a 3-10

membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl

group, a C_3-10 cycloalkynyl group, a thiol group, a cyano group, a hydroxyl

group, or may be a compound having the formula (CH₂)_YNR₈R₉, wherein: (a)

Y is an integer from 1-10; (b) R₈ and R₉ are, independently, hydrogen, an alkyl

group, an alkenyl group, an alkynyl group, an amine, an alkyl amine, a dialkyl

amine, a hydroxy group, an alkoxy group, an acyl group, a carbamate; and (c)

R₈, R₉ and the nitrogen to which they are attached may form a saturated or unsaturated three to ten membered heterocyclic ring.

R⁴ may be a hydrogen, a halogen atom, a hydroxy group, an amino

group, a methoxy group, an alkyl group, an alkynyl group or an alkenyl group.

R⁵ may be hydrogen or fluorine. R⁶ may be an alkyl group, an alkenyl group,

an alkynyl group, or a benzyl group. R⁷ may be a side chain of a naturally

occurring amino acid, or a compound having the formula (CH₂)_LNR¹⁴R¹⁵,

wherein L is an integer ranging from 1-30 and R¹⁴ and R¹⁵ are independently

the same or different and are hydrogen, a C_W5 alkyl group, a C_2-15 alkenyl

group, a C_2-i₅ alkynyl group or an aryl group.

R¹ may be linked to R² in accordance with the structure R¹(CH₂)_QR²,

wherein Q represents an integer 1-10, a SiR_πR₁₂R₁₃ group. Q may also

represent a compound having the formula (CH₂)_FSiR_nR₁₂R₁₃, wherein F is an

integer from 1-10, and R_n, R₁₂ and R₁₃ independently represent hydrogen, a

halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a C₃.₁₀

cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, an

amino group, or a hydroxy group. R² may be linked to R³ in accordance with

the structure R²(CH₂)_GR³, wherein G is an integer from 1-10, and one or more

N, O or S atoms are substituted for one or more -CH₂- groups.

R³ may be linked to R⁴ in accordance with the structure R³(CH₂)_GR⁴

wherein G is an integer from 1-10, and one or more N, O or S atoms are

substituted for one or more -CH₂- groups. Typically, L will be an integer ranging from 1-6.

Suitable camptothecins from which the camptothecin-20-aminoester

derivative may be derived include camptothecins selected from the group

consisting of SN-38, 9-aminocamptothecin, DX-8951f, GG-211, 9-

nitrocamptothecin, topotecan, CPT-11, lurtotecan, CKD-602, 10-

hydroxycamptothecin, and ST 1481.

In another aspect, the present invention provides a method for reducing

toxicity of a camptothecin, comprising the steps of synthesizing a

camptothecin-20-aminoester pro-drug, and incorporating the camptothecin-20-

aminoester pro-drug into an aqueous core of a liposome. The camptothecin-

20-aminoester pro-drug may be selected from compounds having the structure

as described above, and may be synthesized from any camptothecin as

described above.

In yet another aspect, the present invention provides a method for

extending in vivo survival of a camptothecin comprising the steps of

synthesizing a camptothecin-20-aminoester pro-drug having the structure as

described above, and incorporating the camptothecin-20-aminoester pro-drug

into an aqueous core of a liposome. The camptothecin-20-aminoester pro-

drug may be synthesized from the group of camptothecins as described above.

In still yet another aspect of the present invention, a phaπnaceutical

composition is provided, comprising an amine-containing camptothecin derivative incorporated into an aqueous core of a liposome. The amine-

containing camptothecin derivative is typically a camptothecin-20-aminoester

having a structure as described above. The pharmaceutical composition may

be derived from any of the group of camptothecins as described above.

In still yet another aspect of the present invention, a method of forming

a topoisomerase I-inhibiting camptothecin compound in a mammal is

provided, comprising administering a liposome preparation comprising a

liposome containing a camptothecin-20-aminoester derivative in an aqueous

core. The liposome preparation is administered to a mammal in an amount

sufficient to inliibit topoisomerase I. The camptothecin-20-ester may have the

structure as substantially described above, and may be derived from any of the

group of camptothecins as described above. Typically, a sufficient amount of

the liposome preparation of the present invention will be administered to

provide from about 1 to about 200 mg/kg body weight per week of the

camptothecin-20-aminoester derivative. The liposome preparation may be

administered parentally.

In still yet another aspect of the invention, a method of treating cancer

in a mammal, including a human, is provided comprising administering an

effective ^"amount of a liposome preparation. The liposome preparation may

comprise a liposome containing a camptothecin-20-aminoester derivative

having the structure as described above in the aqueous core of the liposome. The camptothecin-20-aminoester derivative may be derived from a

camptothecin selected from the group consisting of SN-38, 9-

aminocamptothecin, DX-8951f, GG-211, 9-nitrocamptothecin, topotecan,

CPT-11, lurtotecan, CKD-602, 10-hydroxycamptothecin, and ST1481 as

described above. Typically, a sufficient amount of the liposome preparation of

the present invention will be administered to provide from about 1 to about

200 mg/kg body weight per week of the camptothecin-20-aminoester

derivative. The liposome preparation may be administered parentally.

Other objects and applications of the present invention will become

apparent to those skilled in this art from the following description wherein

there is shown and described a preferred embodiment of this invention, simply

by way of illustration of the modes currently best suited to carry out the

invention. As it will be realized, the invention is capable of other different

embodiments and its several details are capable of modification in various,

obvious aspects all without departing from the mvention. Accordingly, the

drawings and descriptions will be regarded as illustrative in nature and not as

restrictive.

Brief Description of the Drawings

The accompanying drawing incorporated in and forming a part of the

specification illustrates several aspects of the present invention and, together

with the description, serves to explain the principles of the invention. In the drawing:

Figure 1 shows the structures of clinical candidates and FDA-approved

analogs in the camptothecin family of antitumor agents.

Figure 2 schematically depicts camptothecin hydrolysis at

physiological pH.

Figure 3 shows a structural comparison between SN-38 and DB-67.

Figure 4 schematically depicts the two compartments for drug loading

that are contained within a lipid vesicle. The top panel represents the

situation where an entrapped drug loads predominantly into the lipid bilayer

compartment; the lower panel represents drug loading into the aqueous cavity

contained in the core of the liposomal particle.

Figure 5 shows the mechanism of active drug loading in a liposomal

particle, whereby an amine-containing agent loads into the particle by a

gradient caused by ammonia gas diffusing out of the particle, thereby creating

a driving force for an amine-containing compound to enter the liposome.

Upon being suspended in blood, forces act on the drug and the liposome

promoting release of the agent.

Figure 6 schematically depicts the structures of camptothecin and related camptothecin-20-ester analogs displaying enhanced E-ring lactone stability.

Figure 7 shows a HPLC chromatogram depicting the separation of

camptothecin from camptothecin carboxylate. Figure 8 shows a HPLC chromatogram depicting the markedly

improved solution stability of camptothecin-20-acetate relative to

camptothecin as shown in Figure 7.

Figure 9 illustrates the improved human blood stability of

camptothecin-20-acetate relative to camptothecin in phosphate buffered saline

and in human blood (1 μM drug concentration).

Figure 10 shows HPLC chromatograms depicting the high purity and

high stability in non-aqueous DMSO solution of a hydrochloride salt

preparation (left panel) and a trifluoroacetate salt preparation (right panel) of

camptothecin-20-glycinate ester.

Figure 11 shows HPLC chromatograms depicting the pronounced and

instantaneous reactivity of camptothecin-20-glycinate ester hydrochloride

upon addition to PBS buffer under near physiological conditions of ionic

strength and temperature.

Figure 12 shows HPLC chromatograms depicting the pronounced and

instantaneous reactivity of camptothecin-20-glycinate ester hydrochloride

upon addition to human blood.

Figure 13 illustrates the pronounced reactivity of camptothecin-20-

glycinate ester in human plasma (left panel) and human blood (right panel)

at an initial pro- drug concentration of 1 μM.

Figure 14 shows the impact of pH on the chemical decomposition of camptothecin-20-glycinate ester in PBS buffer at 37 °C at a pro-drug

concentration of 1 μM.

Figure 15 shows the effect of pH on the reversibility of the formation

of the chemical degradation products of camptothecin-20-glycinate ester in

PBS buffer at 37 °C at a pro-drug concentration of 1 μM.

Figure 16 schematically depicts reaction mechanisms which

camptothecin-20-glycinate ester hydrochloride undergoes in aqueous

solution at 37 °C at a pro-drug concentration of 1 μM.

Figure 17 shows stability profiles of several different camptothecin

pro-drug structures in PBS buffer.

Figure 18 shows stability profiles of several different camptothecin

pro-drug structures in whole blood. Data sets are shown for both free drugs

in whole blood as well as core-loaded pro-drugs.

Figure 19 shows normalized fluorescence emission spectra of 1 μM

of camptothecin-20-glycinate ester hydrochloride in PBS buffer at different

pH values.

Figure 20 shows fluorescence excitation and emission spectra of 1

μM of camptothecin-20-glycinate ester hydrochloride in PBS buffer in the

presence and absence of 0.1 M dimyristoylphosphatidyl choline (DMPC) or

0.1 M dimyristoylphosphatidlyglycerol (DMPG) small unilamellar

liposomes. Figure 21 illustrates the associations of camptothecin-20-glycinate

ester to small unilamellar vesicles composed of electroneutral DMPC

(Panel A) and DMPG (Panel B) suspended in phosphate buffered saline,

using fluorescence anisotropy titration.

Figure 22 shows the pronounced reactivity of camptothecin-20-

glycinate ester in the presence of varying concentrations of DMPC SUVs in

PBS buffer at 37 °C at pH 7.4 at an initial pro-drug concentration of 1 μM.

Figure 23 shows the pronounced reactivity of camptothecin-20-

glycinate ester in the presence of varying concentrations of DMPC SUVs in

PBS buffer at 37 °C at pH 10 (top panel) and pH 3.0 at an initial pro-drug

concentration of 1 μM.

Figure 24 illustrates the pronounced stabilization in PBS (left panel)

and whole human blood (right panel) which core loading of camptothecin-

20-glycinate ester hydrochloride into DSPC liposomes provides for pro-

drug stability at 37 °C at pH 7.4.

Figure 25 shows the pronounced stabilization in PBS (left panel) and

whole human blood (right panel) provided by core loading of

camptothecin-20-glycinate ester trifluoroacetate into DSPC liposomes has

on pro-drug stability at 37 °C at pH 7.4.

Figure 26 provides a comparison of the model membrane

associations of several A-ring and A,B-ring modified camptothecin glycinate ester compounds to small unilamellar vesicles (SUVs) composed

of electroneutral dimyristoylphosphatidylcholine (DMPC) and negatively-

charged dimyristoylphosphatidylglycerol (DMPG) in PBS. Data sets are

also shown for camptothecin-20-glycinate ester, 7-chloromethyl-10,l l-

methylenedioxy-camptothecin-20-glycinate ester, 9-chloro- 10,l l-

methylenedioxycampto-thecin-20-glycinate ester, and 7-ethyl-10,l l-

methylenedioxycamptothecin-20-glycinate ester binding to DMPC and

DMPG bilayers.

Figure 27 provides a comparison of the equilibrium binding of

several E-ring modified camptothecin-20-OR esters (where R=CO[CH₂]_n

NH₂HC1 where n= 1-3) to small unilamellar vesicles (SUVs) composed of

electroneutral dimyristoylphosphatidylcholine (DMPC) and negatively-

charged dimyristoylphosphatidylglycerol (DMPG) in PBS. Data sets are also

shown for camptothecin-20-glycinate ester, camptothecin-20-propanate ester,

and camptothecin-20-butanate ester. Data for camptothecin is included for

comparative purposes.

Figure 28 shows the reactivity of highly lipophilic 7-t-

Butyldimethylsilyl-10-hydroxycamptothecin glycinate ester (DB-67

glycinate) in PBS buffer (left panel) and human blood (right panel) at an

initial pro-drug concentration of 1 μM. Figure 29 shows a comparison of the reactivities of several E-ring

modified camptothecin-20-OR esters (where R=CO[CH₂]_nNH₂HCl where n=

1-3) in PBS buffer (left panel) and human blood (right panel) at an initial

pro-drug concentration of 1 μM.

Figure 30 depicts the reactivity of camptothecin-20-propanate ester

hydrochloride in the presence of varying concentrations of DMPC SUVs in

PBS buffer at 37 °C at pH 7.4 at an initial pro-drug concentration of 1 μM.

Figure 31 illustrates the reactivity of camptothecin-20-butanate ester

hydrochloride in the presence of varying concentrations of DMPC SUVs in

PBS buffer at 37 °C at pH 7.4 at an initial pro-drug concentration of 1 μM.

Figure 32 shows the markedly improved stability of core-loaded

liposomal camptothecin-20-propanate in PBS buffer relative to the

corresponding data set for camptothecin-20-glycinate.

Figure 33 summarizes the markedly improved stability of liposomal

core-loaded camptothecin-20-propanate ester in PBS versus bilayer-loaded

camptothecin-20-propanate ester.

Figure 34 shows the markedly improved stability of core-loaded

liposomal camptothecin-20-butanate pro-drug dispersed in human blood

versus drug in its free unencapsulated form.

Figure 35 illustrates the stability of core-loaded liposomal DB-67

glycinate dispersed in human blood. Figure 36 illustrates the stability of DB-67 butanate (free drug) in

PBS buffer and whole human blood.

Figure 37 summarizes the markedly improved stability of core-

loaded liposomal camptothecin-20-glycinate ester in PBS.

Figure 38 shows the markedly improved solution stability of

liposomal DB-67 butanate ester in whole human blood.

Reference will now be made in detail to the presently

preferred embodiments of the invention, examples of which are illustrated

in the accompanying drawing.

Detailed Description of the Invention

In the present patent application we demonstrate how opportunities

in the areas of camptothecin rational design and liposomal formulation can

be used to engineer controlled release vehicles for camptothecins. The

methods and compositions of the present invention may be accomplished by

various means which are illustrated in the examples below. These examples

are intended to be illustrative only, as numerous modifications and

variations will be apparent to those skilled in the art.

We have developed particles that contain camptothecin pro-drugs

that readily core-load, even if the parent active agent is highly lipophilic. This invention provides a means of preparing tumor-targeted liposomes

containing pro-drugs of camptothecin and related analogs.

The simple, versatile and tractable chemically activated pro-drug

(CAP) approach of the present invention can be applied to essentially any

camptothecin including the agents summarized in Figure 1. Many

companies have tried to prepare core-loaded liposomal formulations of

specific camptothecins but have been unable to do so because the active

agent did not possess an amino group of sufficient basic character to allow

for core-loading by active loading methods. Significant but unsuccessful

efforts were made by a number of labs to attempt to core-load 9- AC (an

agent that was evaluated extensively in humans but apparently failed in the

clinic largely because of its highly specific interactions with human albumin

and its resultant poor human blood stability). ALZA and others have been

unable to succeed with generating core-loaded liposomal 9-AC

formulations. Conversion of 9-AC to a CAP (via esterification of a glycine)

should readily allow the agent to core-load. In light of the extensive human

data on the 9-AC agent (trials were terminated at the end of phase III), it

seems logical to examine the potential of liposomal 9-AC CAP. Another

way the CAP approach could find commercial interest is to further optimize

drug retention in the core of liposomes. Gilead has developed GG211 core-

loaded in liposomes; results by this lab and several others have shown that GG211 leaks from the core of NX-211 liposomes. Gilead now has their

core-loaded liposomal GG211 product in phase II clinical trials, but they are

reports of hematological toxicities; these toxicities may be due to premature

leakage of the agent into the blood prior to targeting at the tumor site. Our

CAP approach may have two advantages for the Gilead product: 1) it would

create an inactive prodrug that, in the case of leakage in the blood, would

potentially reduce toxicity; and 2) the additional positive charge earned by

the CAP side-chain may significantly aid in retaining the drug in the particle

until release occurs at the tumor site. CKD602 is now being formulated in

the core of liposomes by ALZA/Johnson and Johnson, and conversion of

CKD602 into a pro-drug may provide for particle retention advantages.

CAP is a versatile approach in that it can be used on any of the

existing camptothecins such as SN-38, 9-AC, 9-NC, and GG-211. The CAP

pro-drug ester approach focuses on position 20(S) of the E-ring and entails

the esterification with the amino acid glycine or larger R=CO[CH₂]_n

NH₂HC1 functionalities. The synthesis of this type of camptothecin pro-

drug ester has been previously described by Wall and Wani (PCT Patent

Application S.N. WO09602546A1) and Vishnuvajjala and Garzon-Aburbeh

(U.S. Patent No. 4,943,579). We have shown that the CAP 20(S)-glycinate

ester is spontaneously converted to the parent agent through a lactam

intermediate. We have shown that the longer chain esters propanate and butanate react at a much slower rate than glycinate (because the resulting

seven- and eight- membered lactams are significantly less stable than the

six-membered lactams formed from glycinate ester decomposition). We

have also demonstrated the pro-drugs are significantly more stable at low

pH, indicating that a low pH environment within the liposomal particle

would be favorable in maintaining the pro-drug intact.

Our specific CAP technology relates to a liposome composition

having entrapped parent camptothecin ester derivatives loaded

predominantly in the aqueous core of the particle. Through judicious

choice of liposomal encapsulation methodologies, lipid ingredients, and the

choice of the R=CO[CH₂]_n NH₂HC1 functionality of the camptothecin-20-

ester, formulations have been described which release camptothecin pro-

drug from the liposomal particle to generate active parent camptothecin in a

rational and controlled fashion resulting in optimal therapeutic efficacy.

Upon departing the liposomal particle at the tumor site the camptothecin -

20-ester undergoes spontaneous decomposition and generates the active

lactone forms of camptothecins which are potent inhibitors of DNA

topoisomerase I and potent anti-cancer agents. Tumor-targeting will allow

for a reduction of systemic toxicity of the encapsulated agent. Thus, the

availability of the liposomal CAP described in this provisional patent

application provides convenient handles on controlling the rate of formation of active camptothecin; using these various delivery systems the optimal

rate of camptothecin parent drug release at the tumor site for optimal

regression to occur can be achieved.

The parent structure camptothecin is water-insoluble and hence a

number of investigators have pursued the development of more water-

soluble agents. U.S. Patent No. 4,943,579 by Vishnuvajjala and Garzon-

Aburbeh discloses water-soluble derivatives of camptothecins having the

formula CPT(20)-OR where R=C0CH(2)NH(2)HC1. U.S. Pat. No.

4,943,579 discloses the esterification of the hydroxyl group at the 20-

position of camptothecin to form several pro-drugs. This patent further

discloses that the pro-drugs are water soluble and are converted into the

parent camptothecin compounds by hydrolysis.

Following the above disclosure by Vishnuvajjala and Garzon-

Aburbeh, Wall and Wani in U.S. Pat. Nos. 5,916,896, 5,646,159, and

6,040,313 disclose that esterification of the hydroxyl group at the 20-

position of camptothecin compounds produces a non-toxic water-soluble

pro-drug. Wall and Wani claim the pro-drug is non-toxic even though the

parent camptothecin compound itself may be substantially more toxic. Wall

and Wani teach hydrolysis of the ester formed at the 20-position reforms the

parent camptothecin compound after administration thereby reducing the

overall toxicity experienced by the patient during camptothecin therapy. Wall and Wani studied the toxicity or non-toxicity of the camptothecin

esters by monitoring weight loss in test animals such as mice which have

been administered the ester compounds. By "non-toxic", Wall and Wani

teach the glycinate ester compounds of camptothecin are not toxic

according to Protocol 4, section 4.301(b)(3) where toxicity is defined as a

weight loss of 4.0 grams as reported in R. I. Geran, N. H. Greenberg, M. M.

MacDonald, A. M. Schumacher and B. J. Abbott, Cancer Chemotherapy

Reports, Part 3, Vol. 3, No. 2, September 1972 (incorporated herein by

reference). Wall and Wani teach that pro-drugs formed by esterifying the

hydroxyl group at the 20-position are non-toxic in contrast to the toxicity of

parent camptothecin compounds even though the esterified derivatives are

hydrolyzed to the parent camptothecin compounds after administration.

Wall and Wani in U.S. Pat. No. 4,943,579 do not suggest that pro-drugs

formed by esterifying the hydroxyl group at the 20-position are non-toxic

relative to the parent compounds, and claim the compounds disclosed in

U.S. Pat. No. 4,943,579 are not within their inventions described in U.S.

Pat. Nos. 5,916,896, 5,646,159, and 6,040,313.

It is important to note that both Vishnuvajjala and Garzon-Aburbeh

and Wall and Wani teach that hydrolysis of the ester formed at the 20-

position reforms the parent camptothecin compound after administration.

Wall and Wani claim that hydrolysis of the exocyclic ester bond in vivo regenerates the parent hydroxyl group containing camptothecin compound.

No additional mechanistic detail is given as to how this hydrolysis occurs.

Alkyl esters have also been described by Cao and Giovanella also contain

an exocyclic ester bond and no information is given by Wall and Wani as to

how the hydrolysis of these two classes of camptothecin esters vary in terms

of the hydrolysis of the exocyclic ester (00=0) bond. Wadkins et al. also

teach that camptothecin glycinate esters also hydrolyze, but no information

is given in their work as to how the exocyclic ester bonds of camptothecin

glycinate esters and camptothecin alkyl esters vary. Further, Wall and Wani

claim in their patent R=CO[CH₂]_nNH₂HCl where n= from a short value of 1

to a long value such as 12 with no discussion whatsoever of how extension

of this chain would impact on hydrolysis. Wall and Wani teach that the

compounds of their invention can be administered in the form of liposome

or microvesicle preparations. They describe liposomes as microvesicles

which encapsulate a liquid within lipid or polymeric membranes.

Liposomes and methods of preparing liposomes are known and are

described by Wall and Wani, for example, in U.S. Pat. No. 4,452,747, U.S.

Pat. No. 4,448,765, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,721,612, U.S.

Pat. No. 4,594,241, U.S. Pat. No. 4,302,459 and U.S. Pat.No. 4,186,183.

The disclosures of these U.S. patents are incorporated herein by reference.

Suitable liposome preparations for use in the present invention are also described in WO-9318749-W, J-02056431-A and EP-276783-A, also

incorporated herein by reference, No mention is made by Wall and Wani as

to how the esters with the R=CO[CH₂]_nNH₂HCl functionality where n= 1

to a long value such as 12 differ in their interactions with liposome

formulations. Further, no discussion is made as to how the esters with the

R=CO[CH₂]_n NH₂HC1 functionality interact with the lipid bilayers of the

liposomal formulations.

In 1992 it was discovered that the active lactone forms of

camptothecins could be readily stabilized by using liposomal carriers (Burke,

U.S. Patents 5,552,156 and 5,736,156, incorporated herein by reference).

Liposomes contain both aqueous and lipid bilayer compartments, and it was

shown that both of these compartments could be utilized to stabilize the

lactone form of the drug. All of the camptothecins studied to date display

some degree of lipophilicity, and some fraction of the drug encapsulated

within a liposomal formulation will be located within the lipid bilayer at any

given time. The bilayer-localized fraction of the camptothecins preferentially

partition as the active lactone form. In this manner the lipid bilayer

compartment of a liposome contributes to stabilizing the active lactone form

of a camptothecin. In 1992 it was also disclosed that agents displaying

reduced lipophilicity could be further stabilized by reducing the pH of the

internal aqueous core (Burke, U.S. Patents 5,552,156 and 5,736,156). The reduced pH of the internal core prevents hydrolysis of drugs localizing in

the internal aqueous compartments and not within the bilayer compartment.

Liposome delivery systems can thus aid in solving an inherent

shortcoming of this important class of anticancer agents by enhancing drug

stability and anticancer activity. The drug-laden liposomal particles can be

administered to patients and prolonged plasma exposure has been achieved.

The particles can be passively or actively targeted to tumor using methods

known in the art and, as a consequence, elevated active lactone levels of the

drug can be realized at this site.

Liposomes can also serve as controlled release depots. As previously

stated, the camptothecins are S-phase specific drags, and it has been shown

that optimal activity is obtained when the tumors of a patient are exposed to

the drugs for continuous periods of time. This approach allows the drug to be

present as the cancer cells cycle through S-phase. Liposomes that target

tumors and slowly release internally contained drugs (such that tumor cells are

continuously exposed to the desired drug) appear to be attractive drug delivery

systems to pursue since they likely provide slow, continuous drug release to

the tumor. The combination of internalizing a non-toxic camptothecin pro-

drug that spontaneously activates at the tumor (at rates controllable by

changing the alkyl linkage length between the ester bond and the amine) and the release determined by the bilayer composition result in novel controlled

release carriers that possess an added advantage of being tumor-targeted.

Example 1

Name: tert-Butoxycarbonylamino-acetic acid 4-ethyl-3,13-dioxo-

3,4, 12, 13-tetrahydro-lH-2-oxa-6, 12fl-diaza-dibenzo[t ,/t]fluoren-4-yl-ester (1)

Molecular Weight: 505 g/mol

Molecular Formula: C₂₇H₂₇N₃0₇

Calculated Log P: 2.3352

Procedure: Camptothecin(CPT) (1.00 g, 2.8 mmol) was placed in an oven

dried flask under Nitrogen. Next anhydrous DMF (25 ml) was added

followed by N-(tert-butoxy-carbonyl)glycine ( 1.06 g, 6.1 mmol), DMAP

(0.189 g, 1.5 mmol) and DCC (1.20g, 5.82 mmol) generating a yellow slurry. o After 5h at 22 C the reaction became clear and the DMF was concentrated

without heating under high vacuum. Next dichloromethane (25x3 ml) was

added and the mixture was filtered each time. Finally, the crude material was

purified by flash chromatography (99:1 CH₂Cl₂/acetone; 97:3 CH₂Cl₂/acetone;

95:5 CH₂Cl₂/acetone; 9:1 CH₂Cl₂/acetone) to provide (1) 482 mg (34% yield)

as a light yellow solid: Η NMR (CD₂C1₂) 300 MHz δ 1.01 (t, J= 7 Hz, 3 H),

1.406 (s, 9 H), 2.03-2.25 (m, 2 H), 4.06 (dd, J,= 18 Hz, J₂= 6 Hz, 1 H), 4.20

(dd,

6 Hz, 1 H), 5.11 (d, J= 19 Hz, 1 H) 5.19 (d, J= 19 Hz, 1

H), 5.30-5.43 (m, 2 H), 5.64 (d, J= 17 Hz, 1 H), 7.26 (s, 1 H), 7.62(t, J= 7 Hz,

1 H), 7.78 (t, J= 7 Hz, 1 H), 7.91 (d, J= 7 Hz, 1 H), 8.14 (d, J= 8 Hz, 1 H),

8.33 (s, IH); ¹³C NMR (CD₂C1₂) 100 MHz δ 8.0, 28.6, 32.0, 43.0, 50.4, 67.4,

77.3, 80.3, 96.4, 120.0, 128.1, 128.5, 128.6, 129.1, 129.8, 130.8, 131.5, 146.0,

146.7, 149.0, 152.7, 156.1, 157.5, 167.6, 170.0.

Example 2

Name: 4-Ethyl-3, 13-dioxo-3,4, 12, 13-tetrahydro- lH-2-oxa-6,12a-diaza-

dibenzo[b,/ϊ]-fluoren-4-yloxycarbonylmethyl-ammonium chloride (2)

Molecular Weight: 442 g/mol

Molecular Formula: C₂₂H₂₀ClN₃O₅

Calculated Log P (on salt w/o counterion): 0.5394

The Boc protected glycinate ester of camptothecin (1) (85 mg, 0.30

mmol) was placed in an oven dried flask. Next a solution of hydrogen

chloride in dioxane (33 ml, 4.0 M) was added dropwise generating a bright

yellow mixture. After 3h the solvent was evaporated, the residue was washed

with ether (3x5 ml) and pumped on overnight providing (2) as a bright yellow

solid weighing 68 mg (92% yield): Η NMR (d₆-DMSO) 400 MHz δ 0.95 (t,

J= 7 Hz, 3 H), 2.06-2.23 (m, 2 H), 4.01-4.06 (m, 1 H), 4.28-4.38 (m,l H),

5.26-5.38 (m, 2 H), 5.55 (s, 2 H), 7.32 (s, 1 H), 7.74 (ddd, J,= 8 Hz, J₂= 7 Hz, J₃= 1 Hz, 1 H), 7.88 (ddd, J,= 8 Hz, J₂= 7 Hz, J₃= 1 Hz, 1 H), 8.15-8.18 (m, 2

H), 8.46-8.62 (br m, 2 H), 8.73 (s, 1 H).

Example 3

Name: 4-Ethyl-3 , 13-dioxo-3 ,4, 12, 13 -tetrahydro- lH-2-oxa-6, 12a-diaza-

dibenzo[b,A]-fluoren-4-yloxycarbonylmethyl-ammonium trifluoroacetate (3)

Molecular Weight: 535 g/mol

Molecular Formula: C₂₅H₂₄F₃N₃0₇

Calculated Log P: 0.5394

Camptothecin ester (1) (20 mg, 0.04 mmol) was placed in an oven

dried flask under nitrogen and anhydrous CH₂C1₂ (0.5 ml) was added. Next o o

TFA (0.5 ml) was added dropwise at 0 C. After 5 h at 22 C the reaction mixture was concentrated providing a light brown oily residue. The residue

was washed with ether (3x1 ml) and placed under high vacuum overnight

providing (3) as a bright yellow solid weighing 21 mg (98% yield): *H NMR

(d₆-DMSO) 400 MHz δ 0.95 (t, J= 7 Hz, 3 H), 2.15-2.22 (m, 2 H), 4.12 (br d,

J= 18 Hz, 1 H), 4.36 (br d, J= 18 Hz, 1 H), 5.25-5.40 (m, 2 H), 5.56 (s, 2 H),

7.30 (s, 1 H), 7.74 (ddd, J_x= 8 Hz, J₂= 7 Hz, J₃= 1 Hz, 1 H), 7.89 (ddd, J_x= 8

Hz, J₂= 7 Hz, J₃= 1 Hz, 1 H), 8.15 (d, J= 1 Hz, 1 H), 8.17 (d, J= 1 Hz, 1 H),

8.34-8.48 (br m, 2 H), 8.74 (s, 1 H); ; LRMS (MALDI) m/z 428 (M+Na), 406

(M+H), 379, 338, 331, 294, 228, 212, 190, 172, 164..

Example 4

Name: 3-tert-Butoxycarbonylamino-propionic acid 4-ethyl-3,13-dioxo-

3,4,12,13-tetrahydro-lH-2-oxa-6,12a-diaza-dibenzo[b, z]fluoren-4-yl ester (4)

Molecular Weight: 519 g/mol

Molecular Formula: C₂₈H₂₉N₃0₇

Calculated Log P: 2.6824

Procedure:

Camptothecin(CPT) (0.50 g, 1.4 mmol) was placed in an oven dried

flask under Nitrogen. Next anhydrous DMF (12.5 ml) was added followed by

3-tert-Butoxycarbonylamino-piOpionic acid (0.55 g, 2.9 mmol), DMAP

(0.087 g, 0.71 mmol) and DCC (0.60 g, 2.9 mmol) generating a yellow slurry. o

After 5h at 22 C the reaction became clear and the DMF was concentrated

without heating under high vacuum. Next dichloromethane (25x3 ml) was

added and the mixture was filtered each time. Finally, the crude material was

purified by flash chromatography (99:1 CH₂Cl₂/acetone; 98:2 CH₂Cl₂/acetone;

96:4 CH₂Cl₂/acetone; 9: 1 CH₂Cl₂/acetone) to provide (4) 306 mg (41% yield)

as a light yellow solid: Η NMR (CD₂C1₂) 400 MHz δ 0.99 (t, J= 7 Hz, 3 H),

1.35 (s, 9 H), 2.00-2.70 (m, 2 H), 2.60-2.80 (m, 2 H), 3.30-3.50 (m, 2 H) 5.10-

5.23 (br s, 1 H), 5.26 (br s, 2 H), 5.37 (d, J= Yl Hz, 1 H), 5.64 (d, J= 17 Hz, 1

H), 7.20 (s, 1 H), 7.67 (ddd, J,= 8 Hz, J₂= 6 Hz, J₃= 1 Hz, 1 H), 7.83 (ddd, J_χ=

8.4 Hz, J₂= 7 Hz, J₃= 2 Hz), 7.97 (d, J= 8 Hz, 1 H), 8.17 (d, J= 8 Hz, 1 H),

8.41 (s, 1 H) ¹³C NMR (CD₂C1₂) 100 MHz δ 8.0, 28.6, 32.0, 35.3, 37.0, 50.5, 67.6, 76.9, 79.5, 96.1, 120.1, 128.4, 128.8, 129.3, 130.0, 131.1, 131.8, 146.4,

147.0, 149.3, 153.0, 156.2, 157.8, 168.3, 172.0; LRMS (El) m/z 519 (M+),

402, 330, 302, 287.

Example 5

Name: 2-(4-Ethyl-3, 13-dioxo-3,4, 12, 13-tetrahydro- lH-2-oxa-6, 12a-

diaza-dibenzo[b,Λ]-fluoren-4-yloxycarbonyl)-ethyl-ammonium chloride (5)

Molecular Weight: 456 g/mol

Molecular Formula: C₂₃H₂₂C1N₃0₅

Calculated Log P(w/o counterion): 0.9040

Camptothecin ester (4) (50 mg, 0.10 mmol) was placed in an oven

dried flask under nitrogen and hydrogen chloride in dioxane (10 ml, 4.0 M)

was added generating a bright yellow solution. After 5 h the dioxane was

concentrated, the residue was washed with ether 3x2 ml and the bright yellow product was placed under high vacuum overnight providing 43.7 mg (96%>

yield) of (5) as the hydrochloride salt: Η NMR (d₆-DMSO) 400 MHz δ 0.92

(t, J= 1 Hz, 3 H), 2.14-2.23 (m, 2 H), 2.94-3.06 (m, 4 H), 5.26-5.38 (m, 2 H),

5.49 (d, J= 17 Hz, 1 H), 5.54 (d, J= 17 Hz, 1 H), 7.17 (s, 1 H), 7.73 (ddd, J_x= 8

Hz, J₂= 7 Hz, J₃= 1 Hz, 1 H), 7.88 (ddd, J= 8 Hz, J₂= 7 Hz, J₃= 1 Hz, 1 H),

7.92-8.08 (br m, 2 H), 8.15 (d, J= 8 Hz, 1 H), 8.17 (d, 8 Hz, 1 H), 8.72 (s, 1

H); HRMS (MALDI) mlz Calcd for C₂₃H₂₁N₃0₅K (M+K) 458.111, found

458.111.

Example 6

Name : 2-(4-Ethyl-3 , 13 -dioxo-3 ,4,12,13 -tetrahydro- lH-2-oxa-6, 12a-

diaza-dibenzo[b,/ϊ]-fluoren-4-yloxycarbonyl)-ethyl-ammoniumtrifluoiOacetate

(6) Molecular Weight: 533 Molecular Formula: C₂₅H₂₂F₃N₃0₇

Calculated Log P(w/o counterion): 0.9040

Camptothecin ester (4) (44.5 mg, 0.09 mmol) was placed in an oven

dried flask under nitrogen and anhydrous CH₂C1₂ (1.0 ml) was added. Next o o TFA (1.0 ml) was added dropwise at 0 C. After 5 h at 22 C the reaction

mixture was concentrated providing a light brown oily residue. The residue

was washed with ether (3x1 ml) and placed under high vacuum overnight

providing (6) as a bright yellow solid weighing 35.2 mg (73% yield): 'H NMR

(d₆-DMSO) 400 MHz δ 0.92 (t, J= 7 Hz, 3 H), 2.10-2.26 (m, 2 H), 2.84-3.10

(m, 4 H), 5.29 (d, J= 20 Hz, 1 H), 5.34 (d, J= 20 Hz, 1 H), 5.49(d, J= 17 Hz, 1

H), 5.54 (d, J= 17 Hz, 1 H), 7.17 (s, 1 H), 7.73 (t, J= 8 Hz, 1 H), 7.78-7.98 (m,

4 H), 8.15 (d, J- 7 Hz, 1 H), 8.17 (d, J= 8 Hz, 1 H), 8.72 (s, 1 H); ¹³C NMR

(CD₂C1₂) 100 MHz δ 7.6, 30.3, 31.1, 34.3, 50.3, 66.5, 76.5, 95.0, 118.9, 127.8,

128.0, 128.7, 128.9, 129.8, 130.6, 131.7, 145.0, 146.1, 147.9, 152.4, 156.5,

167.2, 169.7; LRMS (MALDI) m/z 458 (M+K), 442 (M+Na), 420 (M+H),

331, 228, 212, 164.

Example 7

Name: 4-tert-Butoxycarbonylamino-butyric acid 4-ethyl-3,13-dioxo-

3 ,4, 12, 13-tetrahydro- lH-2-oxa-6, 12a-diaza-dibenzo[t ,/i] fluoren-4-yl ester (7)

Molecular Weight: 533 g/mol

Molecular Formula: C₂₉H₃₁N₃0₇

Calculated Log P: 3.0296

Procedure:

Camptothecin(CPT) (0.50 g, 1.4 mmol) was placed in an oven

dried flask under Nitrogen. Next anhydrous DMF (12.5 ml) was added

followed by 4-tert-Butoxycarbonylamino-butyric acid (0.59 g, 2.9 mmol),

DMAP (0.087 g, 0.71 mmol) and DCC (0.60 g, 2.9 mmol) generating a yellow slurry. After 5h at 22 C the reaction became clear and the DMF was

concentrated without heating under high vacuum. Next dichloromethane

(25x3 ml) was added and the mixture was filtered each time. Finally, the

crude material was purified by flash chromatography (99:1 CH₂Cl₂/acetone;

98:2 CH₂Cl₂/acetone; 96:4 CH₂Cl₂/acetone; 9 : 1 CH₂Cl₂/acetone) to provide (7)

151 mg (20% yield) as a light yellow solid: Η NMR (CD₂C1₂) 400 MHz δ

0.97 (t, J= 7 Hz, 3 H), 1.39 (s, 9 H), 1.82 (p, J= 7 Hz, 2 H), 2.06-2.25 (m, 2

H), 2.52 (t, J= 7 Hz, 2 H), 3.00-3.19 (m, 2 H) 4.94-5.02 (br s, 1 H), 5.19 (d, J=

20 Hz, 1 H), 5.24 (d, J= 20 Hz, 1 H), 5.34 (d, J= 17 Hz, 1 H), 5.60 (d, j= 17

Hz, 1 H), 7.15 (s, 1 H), 7.64 (t, J= 7 Hz, 1 H), 7.80 (t, J- 7 Hz, 1 H), 7.94 (d,

J= 8 Hz, 1 H), 8.18 (d, J= 8 Hz, 1 H), 8.38 (s, 1 H) ¹³C NMR (CD₂C1₂) 100

MHz δ 7.9, 25.6, 28.6, 31.5, 32.0, 40.1, 50.5, 67.5, 76.5, 79.3, 96.1, 120.3,

128.4, 128.4, 128.8, 129.3, 129.9, 131.0, 131.8, 146.5, 146.9, 149.3, 153.0,

156.4, 157.7, 168.1, 172.7; LRMS (El) m/z 533 (M+), 477, 348, 330, 302,

287, 248, 218.

Example 8

Name: 3-(4-Ethyl-3,13-dioxo-3,4,12,13-tetrahydro-lH-2-oxa-6,12a-

diaza-dibenzo[b,/t]-fluoren-4-yloxycarbonyl)-propyl-ammonium chloride (8)

Molecular Weight: 470 g/mol

Molecular Formula: C₂₄H₂₄C1N₃0₅

Calculated Log P w/o counterion: 1.2686

Camptothecin ester (7) (57 mg, 0.11 mmol) was placed in an oven-

dried flask under nitrogen and hydrogen chloride in dioxane (4 ml, 4.0 M) was

added generating a bright yellow solution. After 6 h the dioxane was

concentrated, the residue was washed with ether 3x2 ml and the bright yellow

product was placed under high vacuum overnight providing (8) 48 mg (93%

yield) as the hydrochloride salt: Η NMR (d₆-DMSO) 400 MHz δ 0.89 (t, J= 7

Hz, 3 H), 1.74-1.83 (m, 2 H), 2.06-2.16 (m, 2 H), 2.66 (t, J= 7 Hz, 2 H), 2.74-

2.84 (m, 2 H), 5.28 (s, 2 H), 5.42-5.52 (m, 2 H), 7.04 (s, 1 H), 7.66-7.72 (m, 1 H), 7.80-7.88 (m, 2 ), 8.11 (d, J= 9 Hz, 1 H), 8.13 (d, J= 9 Hz, 1 H), 8.68 (s, 1

H); ¹³C NMR (CD₂C1₂) 100 MHz δ 7.6, 22.2, 30.2, 30.3, 37.7, 50.3, 66.4, 76.0,

94.8, 118.9, 127.9, 128.1, 128.7, 128.9, 129.9, 130.6, 131.8, 145.4, 146.1,

147.9, 152.4, 156.6, 167.4, 171.5; LRMS (MALDI) mlz All (M+K), 456

(M+Na), 434 (M+H).

Example 9

Name: 3-(4-Ethyl-3,13-dioxo-3,4,12,13-tetrahydro-lH-2-oxa-6,12a-

diaza-dibenzo[t3,/ϊ]-fluoren-4-yloxycarbonyl)-proρyl-ammonium trifluoro-

acetate (9)

Molecular Weight: 547 g/mol

Molecular Formula: C₂₆H₂₄F₃N₃0₇

Calculated Log P(w/o counterion): 1.2686 Camptothecin ester (7) (38.4 mg, 0.07 mmol) was placed in an oven-

dried flask under nitrogen and anhydrous CH₂C1₂ (1.0 ml) was added. Next o o

TFA (1.0 ml) was added dropwise at 0 C. After 5 h at 22 C the reaction

mixture was concentrated providing a light brown oily residue. The residue

was washed with ether (3x1 ml) and placed under high vacuum overnight

providing (9) as a bright yellow solid weighing 32.5 mg (85% yield): 'H NMR

(d₆-DMSO) 400 MHz δ 0.93 (t, J= 7 Hz, 3 H), 1.75-1.85 (m, 2 H), 2.10-2.20

(m, 2 H), 2.70 (t, J= 7 Hz, 2 H), 2.80-2.90 (br m, 2 H), 5.26-5.38 (m, 2 H),

5.46-5.56 (m, 2 H), 7.07 (s, 1 H), 7.68-7.82 (m, 3 H), 7.88 (ddd, J_x= 8 Hz, J₂=

7 Hz, J₃= 2 Hz, 1 H), 8.15 (d, J= 8 Hz, 1 H), 8.17 (d, J= 8 Hz, 1 H) 8.72 (s, 1

H);¹³C NMR (CD₂C1₂) 100 MHz δ 7.6, 22.3, 30.1, 30.2, 37.9, 50.3, 66.3, 76.0,

94.7, 118.8, 127.8, 128.1, 128.6, 128.9, 129.9, 130.5, 131.7, 145.3, 146.1,

147.9, 152.3, 156.6, 167.4, 171.5; HRMS (MALDI) mlz Calcd for

C₂₄H₂₄N₃0₅Na(M+Na) 456.1530, found 456.1552.

Example 10

A HPLC chromatogram depicting the separation of camptothecin

(retention time of 6.25 min) from camptothecin carboxylate (retention time of

2.2 min) is shown in Figure 7. Camptothecin samples in PBS buffer were

prepared by adding 1 μM camptothecin from a DMSO stock solution. Note at

a very brief incubation time of 1 min. camptothecin in its lactone form

predominates, but at longer incubation times on the order of several hours the drug has hydrolyzed extensively and its inactive, ring-opened carboxylate

form predominates (right panel). Separation of the lactone and carboxylate

forms of camptothecin was achieved using an isocratic mobile phase

consisting of a mixture of 41% acetonitrile and 59% of the triethylamine

acetate buffer. Both camptothecin forms were detected at an excitation

wavelength of 380 nm and an emission wavelength of 440 nm. A flow rate of

1 mL/min was employed.

A HPLC chromatogram depicting the markedly improved solution

stability of camptothecin-20-acetate (retention time of 7.1 min) relative to

camptothecin (Figure 7) is shown in Figure 8. Note how the camptothecin-20-

acetate sample is stable relative to camptothecin; unlike the parent agent, the

camptothecin-20-acetate analog does not readily convert to a rapidly eluting

carboxylate species upon standing at 37 °C for 3 hr (right panel). Separation

of camptothecin-20-acetate was achieved using an isocratic mobile phase

consisting of a mixture of 41%> acetonitrile to 59% of the triethylamine

acetate buffer. Camptothecin-20-acetate was detected at an excitation

wavelength of 380 nm and an emission wavelength of 440 nm. A flow rate

of 1 mL/min were employed.

Example 11

The improved human blood stability of camptothecin-20-acetate

relative to camptothecin in phosphate buffered saline and in human blood (left panel, 1 μM drug concentration) is shown in Figure 9. While

camptothecin-20-acetate was very stable in PBS and blood with only slight

instability of the agent in the latter matrix being noted at the longer

incubation times, the parent camptothecin agent hydrolyzed rapidly in both

PBS and in human blood. Markedly enhanced human blood stability relative

to camptothecin was also noted for another ester, camptothecin-20-

octanoate (right panel, 1 μM drug concentration). Stability profiles were

determined using HPLC methods. All experiments were conducted at pH

7.4 and 37 °C.

Example 12

HPLC cliromatograms depicting the high purity and high stability in

non-aqueous DMSO solution of a hydrochloride salt preparation (left panel)

and a trifluoroacetate salt preparation (right panel) of camptothecin-20-

glycinate ester are shown in Figure 10. Upon standing in DMSO for hours the

two camptothecin-20-glycinate ester salt forms did not show significant

evidence of hydrolysis or other forms of chemical reactivity. Separation of

camptothecin-20-glycinate ester was achieved using an isocratic mobile

phase consisting of a mixture of 41% acetonitrile to 59% of the

triethylamine acetate buffer. Camptothecin-20-glycinate ester was detected

at an excitation wavelength of 380 nm and an emission wavelength of 440

nm. A flow rate of 1 mL/min was employed. Example 13

HPLC chromatograms depicting the pronounced and instantaneous

reactivity of camptothecin-20-glycinate ester hydrochloride upon addition to

PBS buffer under near physiological conditions of ionic strength and

temperature are shown in Figure 11. The parent camptothecin-20-glycinate

ester peak appears at a retention time of 2.7 min. Immediately following the

addition of camptothecin-20-glycinate ester hydrochloride to PBS buffer at a

concentration of 1 μM, a new peak is observed (retention time of 5.2 min).

Upon further standing, sampling of the drug solution in PBS shows further

decomposition with a total of at least three other chemical entities being

observed in the solution. Separation of starting material (camptothecin-20-

glycinate ester) from the hydrolysis products (camptothecin-20-lactam

intermediate (Retention time of 5.2 min), camptothecin (retention time of

6.3 min) and camptothecin carboxylate (retention time of 2.1 min) was

achieved using an isocratic mobile phase consisting of a mixture of 41%

acetonitrile to 59% of the triethylamine acetate buffer. Camptothecin-20-

glycinate ester and its decomposition products were detected at an

excitation wavelength of 380 nm and an emission wavelength of 440 nm. A

flow rate of 1 mL/min was employed.

Example 14 HPLC chromatograms depicting the pronounced and instantaneous

reactivity of camptothecin-20-glycinate ester hydrochloride upon addition to

human blood are provided in Figure 12. Data are presented for samples

analyzed almost immediately following the addition of 1 μM camptothecin-

20-glycinate ester to human blood (1 minute of incubation, left panel) and at a

longer time of incubation of 3 hr (right panel). The parent camptothecin-20-

glycinate ester peak appears at a retention time of 3.1 min. Immediately

following the addition of camptothecin-20-glycinate ester hydrochloride to

blood at a concentration of 1 μM a new peak was observed (retention time of

6.1 min). Upon further standing to a total incubation time of 3 hours in blood,

HPLC analysis of the sample showed evidence of essentially complete

decomposition. A total of at least three new chemical entities were observed

in the blood suspension to which essentially pure camptothecin-20-glycinate

ester was added. Separation of starting material (camptothecin-20-glycinate

ester) from the reactants [camptothecin-20-lactam intermediate (retention

time of 6.1 min); camptothecin (lactone form, retention time of 7.9 min);

and camptothecin carboxylate (retention time of 3.0 min)] was achieved

using an isocratic mobile phase consisting of a mixture of 41% acetonitrile

to 59%o of the triethylamine acetate buffer. Camptothecin-20-glycinate ester

and its decomposition products were detected at an excitation wavelength of 380 nm and an emission wavelength of 440 nm. A flow rate of 1 mL/min

was employed in the analysis.

Example 15

The pronounced reactivity of camptothecin-20-glycinate ester in

human plasma (left panel) and human blood (right panel) at an initial pro-

drug concentration of 1 μM is shown in Figure 13. Comparison of the

stability of camptothecin-20-glycinate ester in human plasma versus human

blood samples reveals that the agent is initially more reactive in the human

blood sample. In both samples the levels of reaction intermediate

camptothecin-20-lactam were the greatest for the first 30 minutes, and then

during the next 90 minute interval of incubation the carboxylate species

became predominant. Stability profiles were determined using HPLC

methods. All experiments were conducted at 37 °C and sample pH values

were adjusted to 7.4 prior to the initiation of an experiment.

Example 16

Depiction of the impact of pH on the chemical decomposition of

camptothecin-20-glycinate ester in PBS buffer at 37 °C at a pro-drug

concentration of 1 μM are shown in Figure 14. Camptothecin-20-glycinate

ester levels were measured using HPLC analysis. While camptothecin-20-

glycinate ester was relatively stable at a pH value of 3 (with only slight

reactivity observed and 95% of the agent remaining in its original form at an incubation time of 3 hr.), significant decomposition of the agent occurred at

higher pH values. As observed in Figure 11, pronounced and instantaneous

reactivity of camptothecin-20-glycinate ester hydrochloride occurred upon

addition to PBS buffer at pH 7.4 (solid circles).

Example 17

The effect of pH on the reversibility of the formation of the chemical

degradation products of camptothecin-20-glycinate ester in PBS buffer at 37

°C at a pro-drug concentration of 1 μM is shown in Figure 15.

Camptothecin-20-glycinate ester levels and decomposition product levels

were measured using HPLC analysis. As shown in Figure 11, pronounced

and instantaneous reactivity of camptothecin-20-glycinate ester hydrochloride

upon addition to PBS buffer at pH 7.4. Solid triangles represent the levels of

camptothecin-20-lactam intermediate that had formed while open circles

represent the levels of the parent camptothecin-20-glycinate ester pro-drug.

Upon reduction of solution pH to a low value of 2, the data shows that

camptothecin-20-lactam intermediate levels decrease while camptothecin-20-

glycinate ester pro-drug levels become elevated. This data shows that the

lactam intermediate can reform camρtothecin-20-glycinate ester and hence the

reaction is reversible. The formation of the camptothecin-20-glycinate ester

upon pH reduction is consistent with the data contained in Figure 14 indicating

that the parent camptothecin-20-glycinate ester pro-drug predominates at reduced pH values (i.e. at low pH the agent is much more stable to reacting

and forming the lactam intermediate).

Example 18

Reaction mechanisms which camptothecin-20-glycinate ester

hydrochloride undergoes in aqueous solution at 37 °C at a pro-drug

concentration of 1 μM are illustrated in Figure 16. We are the first to

postulate that camptothecin-20-lactam is formed during glycinate ester

degradation. Our novel finding concerning the formation of camptothecin-

20-lactam correlates with the rapid formation of camptothecin lactone and

carboxylate species (versus the much slower formation of lactone and

carboxylate forms released following the hydrolysis of an alkyl ester analog

of camptothecin such as camptothecin-20-acetate or camptothecin-20-

octanoate).

Stability profiles (pH 7.4, 37°C) of several different camptothecin

pro-drug structures in PBS buffer are shown in Figure 17, and demonstrate

the improved stability of the pro-drug structures. Similarly, Figure 18

shows the improved stability of camptothecin pro-drug structures in whole

blood. Data sets are shown for both free drugs in whole blood as well as

core-loaded pro-drugs.

Example 19 The normalized fluorescence emission spectra of 1 μM of

camptothecin-20-glycinate ester hydrochloride in PBS buffer at different pH

values is shown in Figure 19. Note the strong shifting of the emission

spectra to the red region upon increasing solution pH. Experiments were

conducted at 37 °C using exciting light of 370 nm.

Example 20

Figure 20 shows the fluorescence excitation and emission spectra of

1 μM of camptothecin-20-glycinate ester hydrochloride in PBS buffer in the

presence and absence of 0.1 M dimyristoylphosphatidylcholine (DMPC) or

0.1 M dimyristoylphosphatidlyglycerol (DMPG) small unilamellar

liposomes. Experiments were conducted at pH 7.4 and 37°C. Note the

strong shifting of the emission spectra to the blue region or shorter

wavelengths in the presence of liposomes. This spectral shifting is

indicative of drug interaction with the membranes. Experiments were

conducted using exciting light of 370 nm. Spectral parameters of the

various camptothecins and glycinate ester analogs are summarized in Table

13.

Table 1. Fluorescence spectral parameters for Camptothecin

analogues in PBS at pH 7.4

Example 21

Fluorescence anisotropy titration was used to study the associations

of camptothecin-20-glycinate ester to small unilamellar vesicles composed

of electroneutral DMPC (Panel A) and DMPG (Panel B) suspended in

phosphate buffered saline. Results are shown in Figure 21. Experiments

were conducted at pro-drug concentrations of 1 μM at 37 °C using exciting

light of 370 nm. Note that in the presence of increasing amounts of

liposomes the drag anisotropy values increase until the majority of drag is

liposome-bound. Data is also presented in the two panels for camptothecin

for comparative purposes. Analysis of the data reveals that, in the case of

DMPC bilayers, camptothecin-20-glycinate has reduced membrane binding

associations relative to camptothecin both at pH 7.4 and at pH 3.0 (Panel A,

inset). For DMPG bilayers, camptothecin-20-glycinate ester displays

markedly higher affinities due to the electrostatic attractive forces between the positively charged drag and negatively charged membrane. Membrane

association constants for various camptothecins and their glycinate esters

are summarized in Table 2.

Table 2. Association constants for Camptothecin analogues

interacting with unilamellar vesicles of electroneutral DMPC, negatively

charged DMPG in PBS at pH 7.4 and 37°C.

Example 22

Reactivity of camptothecin-20-glycinate ester in the presence of

varying concentrations of DMPC SUVs in PBS buffer at 37 °C at pH 7.4 at

an initial pro-drag concentration of 1 μM is shown in Figure 22.

Comparison of the stability of camptothecin-20-glycinate ester in the

presence of DMPC versus the absence of DMPC reveals that the presence

of membrane initially helps conserve the camptothecin-20 glycinate. Our

novel findings indicate that following several minutes of incubation, the presence of membrane has the net effect of markedly promoting the

conversion of camptothecin-20-glycinate to camptothecin-20-lactam. The

effect of the membranes on promoting levels of camptothecin-20-lactam is

dependent upon the concentration of membrane. Stability profiles were

determined using HPLC methods.

Example 23

Reactivity of camptothecin-20-glycinate ester in the presence of

varying concentrations of DMPC SUVs in PBS buffer at 37 °C at pH 10

(top panel) and pH 3.0 at an initial pro-drag concentration of 1 μM is shown

in Figure 23. Comparison of the stability of camptothecin-20-glycinate

ester in the presence of DMPC versus the absence of DMPC reveals the

presence of membrane initially helps conserve the camptothecin-20

glycinate. Several minutes later the presence of membrane has the net

effect of markedly promoting the conversion of camptothecin-20-glycinate

to camptothecin-20-lactam. The effect of the membranes on promoting

levels of camptothecin-20-lactam is dependent upon the concentration of

membrane. Stability profiles were determined using HPLC methods.

Example 24

Stabilization in PBS (left panel) and whole human blood (right

panel) by core loading of camptothecin-20-glycinate ester hydrochloride

into DSPC liposomes has on pro-drag stability at 37 °C at pH 7.4 is shown in Figure 24. Comparison of the stability profiles for camptothecin-20-

glycinate ester in this figure with data shown in Figure 22 clearly

demonstrate that core loading is much more effective at preventing the

decomposition of camptothecin-20-glycinate ester relative to bilayer

loading. Stability profiles were determined using HPLC methods.

Example 25

Stabilization in PBS (left panel) and whole human blood (right

panel) by core loading of camptothecin-20-glycinate ester trifluoroacetate

into DSPC liposomes has on pro-drag stability at 37 °C at pH 7.4 is shown

in Figure 25. Comparison of the stability profiles for camptothecin-20-

glycinate ester with other data again indicate core loading is much more

effective at preventing the decomposition of camptothecin-20-glycinate

ester relative to bilayer loading. Stability profiles were determined using

HPLC methods.

Example 26

Model membrane associations of several A-ring and A,B-ring

modified camptothecin glycinate ester compounds to small unilamellar

vesicles (SUVs) composed of electroneutral dimyristoylphosphatidylcholine

(DMPC) and negatively-charged dimyristoylphosphatidylglycerol (DMPG) in

PBS are shown in Figure 26. Data are also shown for camptothecin-20-

glycinate ester, 7-chloromethyl-10,l l-methylenedioxy-camptothecin-20- glycinate ester, 9-chloro-10,l l-methylenedioxycampto-thecin-20-glycinate

ester, and 7-ethyl-10,l l-methylenedioxycamptothecin-20-glycinate ester

binding to DMPC and DMPG bilayers. The method of fluorescence anisotropy

titration was used to construct the adsorption isotherms. Experiments were

conducted at drag concentrations of 1 μM in PBS buffer (37 °C). Note how

the anisotropy values vary at a given lipid concentration base upon drag

structure, with certain A-ring and A,B-ring modifications resulting in

significantly more lipophilic agents. Because of the potential for chemical

reactivity of the agents of interest in PBS at pH 7.4, anisotropy values at each

lipid concentration were determined immediately (approx. 30 sec. to 1 min.)

following the addition of the glycinate ester form of each agent to the liposome

so as to minimize loss of the parent glycinate ester prior to sample

measurement. Table 2 summarizes the K values for DMPC as well as DMPG

bilayers.

Example 27

Equilibrium binding of several E-ring modified camptothecin-20-OR

esters (where R=CO[CH₂]_n NH₂HC1 where n= 1-3) to small unilamellar

vesicles (SUVs) composed of electroneutral dimyristoylphosphatidylcholine

(DMPC) and negatively-charged dimyristoylphosphatidylglycerol (DMPG) in

PBS is shown in Figure 27. Data are also shown for camptothecin-20-

glycinate ester, camptothecin-20-propanate ester, and camptothecin-20- butanate ester. Data for camptothecin is included for comparative purposes.

The method of fluorescence anisotropy titration was used to construct the

adsorption isotherms. Experiments were conducted at drug concentrations of 1

μM in PBS buffer (37 °C). Because of the potential for chemical reactivity of

the agents of interest in PBS at pH 7.4, anisotropy values at each lipid

concentration were determined immediately (approx. 30 sec.) following the

addition of the glycinate ester form of each agent to the liposome so as to

minimize loss of the parent ester prior to sample measurement. Table 2

summarizes the K values for DMPC as well as DMPG bilayers.

Example 28

Reactivity of highly lipophilic 7-t-Butyldimethylsilyl-10-

hydroxycamptothecin glycinate ester (DB-67 glycinate) in PBS buffer (left

panel) and human blood (right panel) at an initial pro-drug concentration of

1 μM is shown in Figure 28. In both samples the levels of reaction

intermediate camptothecin-20-lactam were the greatest for the first 30

minutes, and the following 90 minutes of incubation the carboxylate species

became predominant. Stability profiles were determined using HPLC

methods. All experiments were conducted at 37 °C and sample pH values

were adjusted to 7.4 prior to the initiation of an experiment.

Example 29 Reactivities of several E-ring modified camptothecin-20-OR esters

(having the formula R=CO[CH₂]_nNH₂HCl where n= 1-3) in PBS buffer (left

panel) and human blood (right panel) at an initial pro-drug concentration of

1 μM are shown in Figure 29. Note the novel finding depicting how the

stabilities of the compounds increase with increasing n value. The

following order of chemical stability was observed for the agents of interest

in both PBS and whole human blood: camptothecin-20-glycinate ester »

camptothecin-20-propanate ester » camptothecin-20-butanate ester. In the

case of the propanate and butanate ester samples no lactam peak was observed

by HPLC chromatographic analysis indicating the 7-membered and 8-

membered lactam species are far less stable in either PBS solution or blood

suspension relative to the camptothecin-20-lactam (which can be readily

separated), during HPLC chromatographic analysis. Stability profiles were

determined using HPLC methods. All experiments were conducted at 37 °C

and sample pH values were adjusted to 7.4 prior to the initiation of an

experiment.

Example 30

Reactivity of camptothecin-20-propanate ester hydrochloride in the

presence of varying concentrations of DMPC SUVs in PBS buffer at 37 °C

at pH 7.4 at an initial pro-drug concentration of 1 μM is shown in Figure 30.

In dramatic contrast to situation observed for camptothecin-20-glycinate where the presence of lipid bilayer can promote the decomposition of the

the agent, the presence of DMPC clearly aids in stabilizing the

camρtothecin-20-OR ester (where R=CO[CH₂]_nNH₂HCl where n=l for

glycinate and n=2 for propanate). The stabilization effect by DMPC on the

camptothecin-20-propanate is dependent upon the concentration of the lipid

where greater hydrolysis is observed at the lower lipid concentrations.

Stability profiles were determined using HPLC methods.

Example 31

Reactivity of camptothecin-20-butanate ester hydrochloride in the

presence of varying concentrations of DMPC SUVs in PBS buffer at 37 °C

at pH 7.4 at an initial pro-drug concentration of 1 μM is shown in Figure 31.

As observed in the case of camptothecin-20-propanate and in dramatic

contrast to the situation observed for camptothecin-20-glycinate (where

lipid bilayer interactions can promote the decomposition of the agent), the

presence of DMPC aids in stabilizing the camptothecin-20-OR ester (where

R=CO[CH₂]_nNH₂HCl and n=l for glycinate and n=3 for butanate). The

stabilization effect by DMPC on the camptothecin-20-butanate, as observed

in the case of camptothecin-20-propanate, is dependent upon the

concentration of the lipid where greater hydrolysis is observed at the lower

lipid concentrations. Stability profiles were determined using HPLC

methods. Example 32

Improved stability of core-loaded liposomal camptothecin-20-

propanate in PBS buffer relative to the corresponding data set for

camptothecin-20-glycinate is shown in Figure 32. Note at times as long as

3 hrs that liposomal camptothecin-20-propanate remains greater than 98%

intact and unreacted (Panel A), while at times out to 48 hrs greater than 35%

of the liposomal formulation remains as it original pro-drug form (Panel B).

Stability profiles were determined using HPLC methods. All experiments

were conducted at an original pro-drag concentration of 1 μM. Similarly,

improved stability of liposomal core-loaded camptothecin-20-propanate

ester in PBS versus bilayer-loaded is shown in Figure 33.

Example 33

Improved stability of core-loaded liposomal camptothecin-20-

butanate pro-drug dispersed in human blood versus drug in its free

unencapsulated form is shown in Figure 34. Note at times as long as 3 hrs

that liposomal camptothecin-20-butanate remains greater than 98% intact

and unreacted (Panel A). Similarly, Figures 35-38 show improved stability

of core-loaded liposomal DB-67 glycinate (Fig. 35), free DB-67 butanate

(Fig. 36), core-loaded liposomal camρtothecin-20-glycinate ester (Fig. 37)

and liposomal DB-67 butanate ester (Fig. 38) in blood and PBS. The foregoing description of preferred embodiments of the invention

has been presented for purposes of illustration and description. It is not

intended to be exhaustive or to limit the invention to the precise form

disclosed. Obvious modifications or variations are possible in light of the

above teachings. The embodiments were chosen and described to provide

the best illustration of the principles of the invention and its practical

application to thereby enable one of ordinary skill in the art to utilize the

invention in various embodiments and with various modifications as are

suited to the particular use contemplated. All such modifications and

variations are within the scope of the mvention as determined by the

appended claims when interpreted in accordance with the breadth to which

they are fairly, legally and equitably entitled.

Claims

What is claimed is:

1. A composition comprising a camptothecin-20-aminoester

derivative having the structure:

A) wherein R¹ is:

1) hydrogen, a halogen atom, a branched or linear alkyl

group, a branched or linear alkenyl group, a C₃.₇ cycloalkyl group, a

branched or linear alkynyl group, an alkoxy group, an alkylamino group, a

dialkylamino group, an alkylthiol group, a thiol group, a phenyl group, an

amino group, a nitro group, or a cyano group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-10

and R₈ and R₉ are, independently, hydrogen, an alkyl group, an alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine, a

hydroxy group, an alkoxy group, an acyl group, or a carbamate, and (b) R₈,

R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three- to ten-membered heterocyclic ring containing 0, S, and

NR¹⁰ wherein R¹⁰ is a hydrogen, an alkyl group, an alkenyl group, an

alkynyl group, an alkoxy group or a carbamate; or

3) a C ₀ cycloalkyl group, a C ₀ cycloalkenyl group,

or a C_M0 cycloalkynyl group;

B) wherein R² is:

1) hydrogen, a halogen atom, a linear or branched alkyl

group, a linear or branched alkenyl group, a linear or branched alkynyl

group, an amino group, an alkylamino group, a dialkylamino group, a nitro

group, a 3-10 membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀

cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a thiol group, or a cyano

group;

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-

10; and (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, or a carbamate; and (c)

R₈, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring; C) wherein R³ is:

1) hydrogen, a halogen atom, a linear or branched alkyl

group, a linear or branched alkenyl group, a linear or branched alkynyl

group, an amino group, an alkylamino group, a dialkylamino group, a nitro

group, a 3-10 membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀

cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a thiol group, a cyano

group, a hydroxyl group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-

10; (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an alkenyl

group, an alkynyl group, an amine, an alkyl amine, a dialkyl amine, a

hydroxy group, an alkoxy group, an acyl group, a carbamate; and (c) R₈, R₉

and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

D) wherein R⁴ is a hydrogen, a halogen atom, a hydroxy

group, an amino group, a methoxy group, an alkyl group, an alkynyl group

or an alkenyl group;

E) wherein R⁵ is hydrogen or fluorine;

F) wherein R⁶ is an alkyl group, an alkenyl group, an alkynyl

group, or a benzyl group; and

G) wherein R⁷ is: 1) a side chain of a naturally occurring amino acid; or

2) (CH₂)_LNR¹⁴R¹⁵, wherein L is an integer ranging from

1-30 and R¹⁴ and R¹⁵ are independently the same or different and are

hydrogen, a C ₅ alkyl group, a C₂.₁₅ alkenyl group, a C₂.₁₅ alkynyl group or

an aryl group.

2. The composition of claim 1, wherein R¹ is linked to R² in

accordance with the structure R^CTLJ_QR², wherein Q represents an integer

1-10, a SiR_nR₁₂R₁₃ group, or a (CH₂)_FSiR_nR₁₂R₁₃ group, wherein:

A) F is an integer from 1-10; and

B) R_n, R₁₂ and R₁₃ independently represent hydrogen, a

halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a C₃.₁₀

cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, an

amino group, or a hydroxy group.

3. The composition of claim 1, wherein R² is linked to R³ in

accordance with the structure R²(CH₂)_GR³, wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one or

more -CH₂- groups.

4. The composition of claim 1, wherein R³ is linked to R⁴ in

accordance with the structure R³(CH₂)_GR⁴ wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one or

more -CH₂- groups.

5. The composition of claim 1, wherein L is an integer ranging

from 1-6.

6. The composition of claim 1, wherein said camptothecin-20-

aminoester derivative is derived from a camptothecin selected from the

group consisting of SN-38, 9-aminocamptothecin, DX-8951f, GG-211, 9-

nitrocamptothecin, topotecan, CPT-11, lurtotecan, CKD-602, 10-

hydroxycamptothecin, and ST 1481.

7. A method for reducing toxicity of a camptothecin, comprising

the steps of:

synthesizing a camptothecin-20-aminoester pro-drug;

and

incorporating said camptothecin-20-aminoester pro-

drag into an aqueous core of a liposome.

8. The method of claim 7, wherein said camptothecin-20-

aminoester pro-drag is a camptothecin derivative having the structure:

A) wherein R¹ is:

1) hydrogen, a halogen atom, a branched or linear alkyl

group, a branched or linear alkenyl group, a C₃.₇ cycloalkyl group, a

branched or linear alkynyl group, an alkoxy group, an alkylamino group, a

dialkylamino group, an alkylthiol group, a thiol group, a phenyl group, an

amino group, a nitro group, or a cyano group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-10

and R₈ and R₉ are, independently, hydrogen, an alkyl group, an alkenyl

group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine, a

hydroxy group, an alkoxy group, an acyl group, or a carbamate, and (b) R₈, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three- to ten-membered heterocyclic ring containing 0, S, and

NR¹⁰ wherein R¹⁰ is a hydrogen, an alkyl group, an alkenyl group, an

alkynyl group, an alkoxy group or a carbamate; or

3) a C ₀ cycloalkyl group, a C_M0 cycloalkenyl group,

or a C ₀ cycloalkynyl group;

B) wherein R² is:

1) hydrogen, a halogen atom, a linear or branched alkyl

group, a linear or branched alkenyl group, a linear or branched alkynyl

group, an amino group, an alkylamino group, a dialkylamino group, a nitro

group, a 3-10 membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀

cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a thiol group, or a cyano

group;

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-

10; and (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, or a carbamate; and (c)

R₈, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

C) wherein R³ is: 1) hydrogen, a halogen atom, a linear or branched alkyl

group, a linear or branched alkenyl group, a linear or branched alkynyl

group, an amino group, an alkylamino group, a dialkylamino group, a nitro

group, a 3-10 membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀

cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a thiol group, a cyano

group, a hydroxyl group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-

10; (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an alkenyl

group, an alkynyl group, an amine, an alkyl amine, a dialkyl amine, a

hydroxy group, an alkoxy group, an acyl group, a carbamate; and (c) R₈, R₉

and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

D) wherein R⁴ is a hydrogen, a halogen atom, a hydroxy

group, an amino group, a methoxy group, an alkyl group, an alkynyl group

or an alkenyl group;

E) wherein R⁵ is hydrogen or fluorine;

F) wherein R⁶ is an alkyl group, an alkenyl group, an alkynyl

group, or a benzyl group; and

G) wherein R⁷ is:

1) a side chain of a naturally occurring amino acid; or 2) (CH₂)_LNR¹⁴R¹⁵, wherein L is an integer ranging from

1-30 and R¹⁴ and R¹⁵ are independently the same or different and are

hydrogen, a C ₅ alkyl group, a C_2-15 alkenyl group, a C_2-15 alkynyl group or

an aryl group.

9. The method of claim 8, wherein R¹ is linked to R² in

accordance with the stracture R^CH^_QR², wherein Q represents an integer

1-10, a SiR_nR₁₂R₁₃ group, or a (CH₂)_FSiR_nR₁₂R₁₃ group, wherein:

A) F is an integer from 1-10; and

B) R_u, R₁₂ and R₁₃ independently represent hydrogen, a

halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a C₃.₁₀

cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, an

amino group, or a hydroxy group.

10. The method of claim 8, wherein R² is linked to R³ in

accordance with the stracture R²(CH₂)_GR³, wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one or

more -CH₂- groups.

11. The method of claim 8, wherein R³ is linked to R⁴ in

accordance with the structure R³(CH₂)_GR⁴ wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one or

more -CH₂- groups.

12. The method of claim 8, wherein L is an integer ranging from

1-6.

13. The method of claim 8, wherein said camptothecin-20-

aminoester derivative is derived from a camptothecin selected from the

group consisting of SN-38, 9-aminocamptothecin, DX-8951f, GG-211, 9-

nitrocamptothecin, topotecan, CPT-11, lurtotecan, CKD-602, 10-

hydroxycamptothecin, and ST 1481.

14. A method for extending in vivo survival of a camptothecin

comprising the steps of:

synthesizing a camptothecin-20-aminoester pro-drug;

and

incorporating said camptothecin-20-aminoester pro-

drag into an aqueous core of a liposome.

15. The method of claim 14, wherein said camptothecin-20-

aminoester pro-drag is a camptothecin derivative having the structure:

A) wherein R¹ is:

1) hydrogen, a halogen atom, a branched or linear alkyl

group, a branched or linear alkenyl group, a C₃.₇ cycloalkyl group, a

branched or linear alkynyl group, an alkoxy group, an alkylamino group, a

dialkylamino group, an alkylthiol group, a thiol group, a phenyl group, an

amino group, a nitro group, or a cyano group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-10

and R₈ and R₉ are, independently, hydrogen, an alkyl group, an alkenyl

group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine, a

hydroxy group, an alkoxy group, an acyl group, or a carbamate, and (b) R₈, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three- to ten-membered heterocyclic ring containing O, S, and

NR¹⁰ wherein R¹⁰ is a hydrogen, an alkyl group, an alkenyl group, an

alkynyl group, an alkoxy group or a carbamate; or

3) a C ₀ cycloalkyl group, a C ₀ cycloalkenyl group,

or a C,.₁₀ cycloalkynyl group;

B) wherein R² is:

1) hydrogen, a halogen atom, a linear or branched alkyl

group, a linear or branched alkenyl group, a linear or branched alkynyl

group, an amino group, an alkylamino group, a dialkylamino group, a nitro

group, a 3-10 membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀

cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a thiol group, or a cyano

group;

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-

10; and (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, or a carbamate; and (c)

R₈, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

C) wherein R³ is: 1) hydrogen, a halogen atom, a linear or branched alkyl

group, a linear or branched alkenyl group, a linear or branched alkynyl

group, an amino group, an alkylamino group, a dialkylamino group, a nitro

group, a 3-10 membered heterocyclic ring, a C₃.₁₀ cycloalkyl group, a C₃.₁₀

cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a thiol group, a cyano

group, a hydroxyl group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer from 1-

10; (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an alkenyl

group, an alkynyl group, an amine, an alkyl amine, a dialkyl amine, a

hydroxy group, an alkoxy group, an acyl group, a carbamate; and (c) R₈, R₉

and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

D) wherein R⁴ is a hydrogen, a halogen atom, a hydroxy

group, an amino group, a methoxy group, an alkyl group, an alkynyl group

or an alkenyl group;

E) wherein R⁵ is hydrogen or fluorine;

F) wherein R⁶ is an alkyl group, an alkenyl group, an alkynyl

group, or a benzyl group; and

G) wherein R⁷ is:

1) a side chain of a naturally occurring amino acid; or 2) (CH₂)_LNR¹⁴R¹⁵, wherein L is an integer ranging from

1-30 and R¹⁴ and R¹⁵ are independently the same or different and are

hydrogen, a C_M5 alkyl group, a C₂.₁₅ alkenyl group, a C₂.₁₅ alkynyl group or

an aryl group.

16. The method of claim 15, wherein R¹ is linked to R² in

accordance with the stracture R^CH^_QR², wherein Q represents an integer

1-10, a SiR_nRι₂R₁₃ group, or a (CH₂)_FSiR_nR₁₂R₁₃ group, wherein:

A) F is an integer from 1-10; and

B) R_n, R₁₂ and R₁₃ independently represent hydrogen, a

halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a C₃.₁₀

cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, an

amino group, or a hydroxy group.

17. The method of claim 15, wherein R² is linked to R³ in

accordance with the structure R²(CH₂)_GR³, wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one or

more -CH₂- groups.

18. The method of claim 15, wherein R³ is linked to R⁴ in

accordance with the stracture R³(CH₂)_GR⁴ wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one or

more -CH₂- groups.

19. The method of claim 15, wherein L is an integer ranging from

1-6.

20. The method of claim 15, wherein said camptothecin-20-

aminoester derivative is derived from a camptothecin selected from the

group consisting of SN-38, 9-aminocamptothecin, DX-8951f, GG-211, 9-

nitrocamptothecin, topotecan, CPT-11, lurtotecan, CKD-602, 10-

hydroxycamptothecin, and ST1481.

21. A pharmaceutical composition comprising an amine-

containing camptothecin derivative incorporated into an aqueous core of a

liposome.

22. The composition of claim 21, wherein said camptothecin

derivative is a camρtothecin-20-aminoester having the stracture:

A) wherein R¹ is:

1) hydrogen, a halogen atom, a branched or

linear alkyl group, a branched or linear alkenyl group, a C₃.₇ cycloalkyl

group, a branched or linear alkynyl group, an alkoxy group, an alkylamino

group, a dialkylamino group, an alkylthiol group, a thiol group, a phenyl

group, an amino group, a nitro group, or a cyano group; or

2) (CH₂)_yNR₈R₉, wherein: (a) Y is an integer

from 1-10 and R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, or a carbamate, and (b)

R₈, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three- to ten-membered heterocyclic ring containing 0, S, and NR¹⁰ wherein R¹⁰ is a hydrogen, an alkyl group, an alkenyl group, an

alkynyl group, an alkoxy group or a carbamate; or

3) a C ₀ cycloalkyl group, a C_M0 cycloalkenyl

group, or a C_M0 cycloalkynyl group;

B) wherein R² is:

1) hydrogen, a halogen atom, a linear or

branched alkyl group, a linear or branched alkenyl group, a linear or

branched alkynyl group, an amino group, an alkylamino group, a

dialkylamino group, a nitro group, a 3-10 membered heterocyclic ring, a C₃.

₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a

thiol group, or a cyano group;

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10; and (b) R₈ and R₉ are, independently, hydrogen, an alkyl group,

an alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl

amine, a hydroxy group, an alkoxy group, an acyl group, or a carbamate;

and (c) R₈, R₉ and the nitrogen to which they are attached may form a

saturated or unsaturated three to ten membered heterocyclic ring;

C) wherein R³ is:

1) hydrogen, a halogen atom, a linear or

branched alkyl group, a linear or branched alkenyl group, a linear or

branched alkynyl group, an amino group, an alkylamino group, a dialkylamino group, a nitro group, a 3-10 membered heterocyclic ring, a C₃_

₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C_3-10 cycloalkynyl group, a

thiol group, a cyano group, a hydroxyl group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10; (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group, an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, a carbamate; and (c) R₈,

R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

D) wherein R⁴ is a hydrogen, a halogen atom, a

hydroxy group, an amino group, a methoxy group, an alkyl group, an

alkynyl group or an alkenyl group;

E) wherein R⁵ is hydrogen or fluorine;

F) wherein R⁶ is an alkyl group, an alkenyl group, an

alkynyl group, or a benzyl group; and

G) wherein R⁷ is:

1) a side chain of a naturally occurring amino

acid; or

2) (CH₂)_LNR¹⁴R¹⁵, wherein L is an integer

ranging from 1-30 and R¹⁴ and R¹⁵ are independently the same or different and are hydrogen, a C_M5 alkyl group, a C_2-15 alkenyl group, a C₂.₁₅ alkynyl

group or an aryl group.

23. The composition of claim 22, wherein R¹ is linked to R² in

accordance with the structure R^CH^_QR², wherein Q represents an integer

1-10, a SiR_nRι₂R₁₃ group, or a (CH₂)_FSiR_nR₁₂R₁₃ group, wherein:

A) F is an integer from 1-10; and

B) R_n, R₁₂ and R₁₃ independently represent hydrogen, a

halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a C₃.₁₀

cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, an

amino group, or a hydroxy group.

24. The composition of claim 22, wherein R² is linked to R³ in

accordance with the structure R²(CH₂)_GR³, wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one

or more -CH₂- groups.

25. The composition of claim 22, wherein R³ is linked to R⁴ in

accordance with the structure R³(CH₂)_GR⁴ wherein:

A) G is an integer from 1-10; and B) one or more N, O or S atoms are substituted for one

or more -CH₂- groups.

26. The composition of claim 22, wherein L is an integer ranging

from 1-6.

27. The composition of claim 22, wherein said camptothecin-20-

aminoester derivative is derived from a camptothecin selected from the

group consisting of SN-38, 9-aminocamptothecin, DX-8951f, GG-211, 9-

nitrocamptothecin, topotecan, CPT-11, lurtotecan, CKD-602, 10-

hydroxycamptothecin, and ST1481.

28. A method of forming a topoisomerase I-inhibiting

camptothecin compound in a mammal, comprising administering a

liposome preparation comprising a liposome containing a camptothecin-20-

aminoester derivative in an aqueous core, said liposome preparation being

administered to said mammal in an amount sufficient to inhibit

topoisomerase I.

29. The method of claim 28, wherein said camptothecin-20-

aminoester derivative has the structure:

A) wherein R¹ is:

1) hydrogen, a halogen atom, a branched or

linear alkyl group, a branched or linear alkenyl group, a C₃.₇ cycloalkyl

group, a branched or linear alkynyl group, an alkoxy group, an alkylamino

group, a dialkylamino group, an alkylthiol group, a thiol group, a phenyl

group, an amino group, a nitro group, or a cyano group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10 and R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, or a carbamate, and (b)

R₈, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three- to ten-membered heterocyclic ring containing 0, S, and NR¹⁰ wherein R¹⁰ is a hydrogen, an alkyl group, an alkenyl group, an

alkynyl group, an alkoxy group or a carbamate; or

3) a C ₀ cycloalkyl group, a C ₀ cycloalkenyl

group, or a C ₀ cycloalkynyl group;

B) wherein R² is:

1) hydrogen, a halogen atom, a linear or

branched alkyl group, a linear or branched alkenyl group, a linear or

branched alkynyl group, an amino group, an alkylamino group, a

dialkylamino group, a nitro group, a 3-10 membered heterocyclic ring, a C₃_

₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a

thiol group, or a cyano group;

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10; and (b) R₈ and R₉ are, independently, hydrogen, an alkyl group,

an alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl

amine, a hydroxy group, an alkoxy group, an acyl group, or a carbamate;

and (c) R₈, R₉ and the nitrogen to which they are attached may form a

saturated or unsaturated three to ten membered heterocyclic ring;

C) wherein R³ is:

1) hydrogen, a halogen atom, a linear or

branched alkyl group, a linear or branched alkenyl group, a linear or

branched alkynyl group, an amino group, an alkylamino group, a dialkylamino group, a nitro group, a 3-10 membered heterocyclic ring, a C₃_

₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a

thiol group, a cyano group, a hydroxyl group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10; (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group, an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, a carbamate; and (c) R₈,

R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

D) wherein R⁴ is a hydrogen, a halogen atom, a

hydroxy group, an amino group, a methoxy group, an alkyl group, an

alkynyl group or an alkenyl group;

E) wherein R⁵ is hydrogen or fluorine;

F) wherein R⁶ is an alkyl group, an alkenyl group, an

alkynyl group, or a benzyl group; and

G) wherein R⁷ is:

1) a side chain of a naturally occurring amino

acid; or

2) (CH₂)_LNR¹⁴R¹⁵, wherein L is an integer

ranging from 1-30 and R¹⁴ and R¹⁵ are independently the same or different and are hydrogen, a C,.₁₅ alkyl group, a C₂.₁₅ alkenyl group, a C₂.₁₅ alkynyl

group or an aryl group.

30. The method of claim 29, wherein R¹ is linked to R² in

accordance with the structure R^CH^_QR², wherein Q represents an integer

1-10, a SiR_nR₁₂R₁₃ group, or a (CH₂)_FSiR_uR₁₂R₁₃ group, wherein:

A) F is an integer from 1-10; and

B) R_π, R₁₂ and R₁₃ independently represent hydrogen, a

halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a C₃.₁₀

cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃„₁₀ cycloalkynyl group, an

amino group, or a hydroxy group.

31. The method of claim 29, wherein R² is linked to R³ in

accordance with the structure R²(CH₂)_GR³, wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one

or more -CH₂- groups.

32. The method of claim 29, wherein R³ is linked to R⁴ in

accordance with the structure R³(CH₂)_GR⁴ wherein:

A) G is an integer from 1-10; and B) one or more N, O or S atoms are substituted for one

or more -CH₂- groups.

33. The method of claim 29, wherein L is an integer ranging from

1-6.

34. The method of claim 29, wherein said camptothecin-20-

aminoester derivative is derived from a camptothecin selected from the

group consisting of SN-38, 9-aminocamptothecin, DX-8951f, GG-211, 9-

nitrocamptothecin, topotecan, CPT-11, lurtotecan, CKD-602, 10-

hydroxycamptothecin, and ST 1481.

35. The method of claim 28, comprising administering sufficient

liposome preparation to provide from about 1 to about 200 mg/kg body

weight per week of the camptothecin-20-aminoester derivative .

36. The method of claim 28, wherein said liposome preparation is

administered parenterally.

37. A method of treating cancer in a mammal, comprising

administering an effective amount of a liposome preparation comprising a liposome containing a camptothecin-20-aminoester derivative in an aqueous

core.

38. The method of claim 37, wherein said camptothecin-20-

aminoester derivative has the structure:

A) wherein R¹ is:

1) hydrogen, a halogen atom, a branched or

linear alkyl group, a branched or linear alkenyl group, a C_3.7 cycloalkyl

group, a branched or linear alkynyl group, an alkoxy group, an alkylamino

group, a dialkylamino group, an alkylthiol group, a thiol group, a phenyl

group, an amino group, a nitro group, or a cyano group; or 2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10 and R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, or a carbamate, and (b)

R_g, R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated tliree- to ten-membered heterocyclic ring containing O, S, and

NR¹⁰ wherein R¹⁰ is a hydrogen, an alkyl group, an alkenyl group, an

alkynyl group, an alkoxy group or a carbamate; or

3) a C ₀ cycloalkyl group, a C_x._xo cycloalkenyl

group, or a C ₀ cycloalkynyl group;

B) wherein R² is:

1) hydrogen, a halogen atom, a linear or

branched alkyl group, a linear or branched alkenyl group, a linear or

branched alkynyl group, an amino group, an alkylamino group, a

dialkylamino group, a nitro group, a 3-10 membered heterocyclic ring, a C₃_

₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a

thiol group, or a cyano group;

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10; and (b) R₈ and R₉ are, independently, hydrogen, an alkyl group,

an alkenyl group , an alkynyl group, an amine, an alkyl amine, a dialkyl

amine, a hydroxy group, an alkoxy group, an acyl group, or a carbamate; and (c) R₈, R₉ and the nitrogen to which they are attached may form a

saturated or unsaturated three to ten membered heterocyclic ring;

C) wherein R³ is:

1) hydrogen, a halogen atom, a linear or

branched alkyl group, a linear or branched alkenyl group, a linear or

branched alkynyl group, an amino group, an alkylamino group, a

dialkylamino group, a nitro group, a 3-10 membered heterocyclic ring, a C₃.

₁₀ cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, a

thiol group, a cyano group, a hydroxyl group; or

2) (CH₂)_YNR₈R₉, wherein: (a) Y is an integer

from 1-10; (b) R₈ and R₉ are, independently, hydrogen, an alkyl group, an

alkenyl group, an alkynyl group, an amine, an alkyl amine, a dialkyl amine,

a hydroxy group, an alkoxy group, an acyl group, a carbamate; and (c) R₈,

R₉ and the nitrogen to which they are attached may form a saturated or

unsaturated three to ten membered heterocyclic ring;

D) wherein R⁴ is a hydrogen, a halogen atom, a

hydroxy group, an amino group, a methoxy group, an alkyl group, an

alkynyl group or an alkenyl group;

E) wherein R⁵ is hydrogen or fluorine;

F) wherein R⁶ is an alkyl group, an alkenyl group, an

alkynyl group, or a benzyl group; and G) wherein R⁷ is:

1) a side chain of a naturally occurring amino

acid; or

2) (CH₂)_LNR¹⁴R¹⁵, wherein L is an integer

ranging from 1-30 and R¹⁴ and R¹⁵ are independently the same or different

and are hydrogen, a C_M5 alkyl group, a C₂.₁₅ alkenyl group, a C₂.₁₅ alkynyl

group or an aryl group.

39. The method of claim 38, wherein R¹ is linked to R² in

accordance with the stracture R'(CH₂)_QR², wherein Q represents an integer

1-10, a SiR_nR₁₂R₁₃ group, or a (CH₂)_FSiR_nR₁₂R₁₃ group, wherein:

A) F is an integer from 1-10; and

B) R_n, Ri₂ and R₁₃ independently represent hydrogen, a

halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a C_3.10

cycloalkyl group, a C₃.₁₀ cycloalkenyl group, a C₃.₁₀ cycloalkynyl group, an

amino group, or a hydroxy group.

40. The method of claim 38, wherein R² is linked to R³ in

accordance with the structure R²(CH₂)_GR³, wherein:

A) G is an integer from 1-10; and B) one or more N, O or S atoms are substituted for one

or more -CH₂- groups.

41. The method of claim 38, wherein R³ is linked to R⁴ in

accordance with the structure R³(CH₂)_GR⁴ wherein:

A) G is an integer from 1-10; and

B) one or more N, O or S atoms are substituted for one

or more -CH₂- groups.

42. The method of claim 38, wherein L is an integer ranging from

1-6.

43. The method of claim 38, wherein said camptothecin-20-

aminoester derivative is derived from a camptothecin selected from the

group consisting of SN-38, 9-aminocamptothecin, DX-8951f, GG-211, 9-

nitrocamptothecin, topotecan, CPT-11, lurtotecan, CKD-602, 10-

hydroxycamptothecin, and ST1481.

44. The method of claim 37, comprising administering sufficient

liposome preparation to provide from about 1 to about 200 mg/kg body

weight per week of the camptothecin-20-aminoester derivative.

45. The method of claim 37, wherein said liposome preparation is

administered parenterally.

46. The method of claim 37, wherein said mammal is a human.