FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
PROVISIONAL SPECIFICATION
(SECTION 10)
INDIVIDIUAL DIASTEREOMERIC C-5 SUBSTITUTED
ANALOGUE OF 20(S)-CAMPTOTHECIN
Dr. Reddy's Laboratories Ltd.,
an Indian Company having its registered office at
7-1-27, Ameerpet
Hyderabad - 500 016, Andhra Pradesh, India
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION:
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FIELD OF THE INVENTION
The present invention relates to analogues of 20(S)-camptothecin, and more particularly to C-5 substituted analogues of 20(S)-camptothecin.
BACKGROUND OF THE INVENTION
Camptothecin (CPT) is a plant alkaloid first isolated from the Chinese tree Camptotheca acuminate. CPT exhibits tumor cell cytotoxicity through topoisomerase I inhibition. Various C-5 substituted analogues of 20 (S)-camptothecin are described in U.S. Patent No. 6,177,439, the content of which is specifically incorporated herein by reference in its entirety. One such analogue, 5(RS)-(2'-hydroxyethoxy)-20(S)-camptothecin of the structure:

is among the substances described in U.S. Patent No. 6,177,439 as a mixture of 5 (R) and 5(S) diastereomers (5(RS)-camptothecins) . The V439 patent does not disclose individual 5(R) and 5(S) diastereomers of 5- (2 '-hydroxyethoxy)-20(S)-camptothecin. Likewise, neither individual diastereomers substantially free of each other nor methods for their preparation or use are described.
SUMMARY OF THE INVENTION
In one aspect, there is provided a compound, which is 5 (S) -(2'-hydroxyethoxy)-20(S)-camptothecin having the structure
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The compound being substantially free from 5(R)- (2 ' -hydroxyethoxy)-20(S)-campthothecin. In a variant of this aspect, the compound is substantially optically pure. In an embodiment of this aspect, the compound has overall chemical purity of over 98%. It is preferred that the amount of 5(R)-(2'-hydroxyethoxy)-20(S)-campthothecin in the compound of this aspect is less than about 1.5% w/w; more preferred, less than about 1% w/w; still more preferred, less than about 0.5% w/w; yet more preferred, less than 0.1% w/w.
In another aspect, there is provided 5 (S)- (2'-hydroxyethoxy)-20 (S)-camptothecin, which is in crystalline form as shown below. In a variant, the crystalline form has a powder X-ray diffraction pattern that includes five or more peaks selected from the group
consisting of 4.6 ± 0.1, 7.2 ± 0.1, 9.4 ± 0.1, 11.0 ± 0.1, 12.0 ±
0.1, 14.1 ± 0.1, 14.5 ± 0.1, 15.2 ± 0.1, 16.2 ± 0.1, 17.1 ± 0.1,
17.9 ± 0.1, 18.9 ± 0.1, 19.1 ± 0.1, 19.7 ± 0.1, 21.2 ± 0.1, 22.7 ±
0.1, 23.2 ± 0.1, 23.7 ± 0.1, 24.2 ± 0.1, 24.4 ± 0.1, 25.3 ± 0.1,
26.4 ± 0.1, 27.1 ± 0.1, 28.6 ± 0.1, 29.1 ± 0.1, 29.4 ± 0.1, 30.1 ±
0.1, 31.0 ± 0.1, 31.4 ± 0.1, 31.9 ± 0.1, 32.5 ± 0.1, 32.7 ± 0,1,
33.9 ± 0.1, 35.1 ± 0.1, 36.7 ± 0.1, 37.6 ± 0.1, 38.2 ± 0.1, 40.3 ±
0.1, 42.1 ± 0.1, 42.2 ± 0.1, and 44.0 ± 0.1 degrees 20, In one embodiment, the crystalline form of 5 (S)-(2'-hydroxyethoxy)-20(S) -camptothecin is characterized by an X-ray diffraction pattern substantially as shown in FIG. 2. In one embodiment, the crystalline form of 5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin is characterized by a DSC thermogram substantially as shown in FIG. 4. Anhydrous form of the compound is preferred.
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In another aspect, there is provided a pharmaceutical composition for oral administration comprising 5 (S)- (2'-hydroxyethoxy)-20(S)-camptothecin and one or more pharmaceutically acceptable carriers. The pharmaceutical composition of this aspect may be in unit dosage form, such as a tablet or a capsule.
In another aspect, there is provided a process for preparing 5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin comprising the steps of:
a) providing a solution and/or suspension of 5(RS)-(2’-hydroxyethoxy)-20(S)-camptothecin in an organic solvent in which said 5(S) -(2'-hydroxyethoxy)-20(S)-camptothecin has lower solubility than said 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin;
b)heating said solution and/or suspension;
c) cooling said solution and/or suspension so that a precipitate is formed; and
d)isolating the precipitate to yield said 5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R) -(2'-hydroxyethoxy)-20(S)-camptothecin.
Preferred organic solvents include n-butanol and THF.
The process of this aspect may include the heating step that comprises refluxing said solution and/or suspension. The process of this aspect may further comprise refluxing the isolated precipitate with additional portions of said organic solvent.
In another aspect, there is provided a method for inhibiting topoisomerase activity in a cell comprising administering to said cell an effective amount of 5(S)-(2 r-hydroxyethoxy)-20(S)-camptothecin which is substantially free from 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin. The target cell may be cancerous and the administration may be carried out in vitro or in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows an HPLC chromatogram of one sample of 5 (S) - (2' -hydroxyethoxy)-20(S)-camptothecin.
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FIG. IB shows an HPLC chromatogram of another sample of 5 (S)- (2'-
hydroxyethoxy)-20(S)-camptothecin.
FIG. 2 shows the X-ray powder diffraction pattern of one batch of
solid powder of 5(S)-(2’-hydroxyethoxy)-20(S)-camptothecin.
FIG. 3A shows the X-ray powder diffraction pattern of one batch of
solid powder of 5-(2'-hydroxyethoxy)-20(S)-camptothecin.
FIG. 3B shows the X-ray powder diffraction pattern of one batch of
solid powder of 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin.
FIG. 4 shows the DSC thermogram of one batch of solid powder of
5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin.
FIG. 5A shows the DSC thermogram of one batch of solid powder of
5-(2'-hydroxyethoxy)-20(S)-camptothecin.
FIG. 5B shows the DSC thermogram of 5(R)-(2!-hydroxyethoxy)-20(S)-
camptothecin.
DETAILED DESCRIPTION OF THE INVENTION
To describe the invention, certain terms are defined herein as follows.
The use of singular includes the use of plural. In a non-limiting example, a recitation of "a compound" includes a single molecule, as well as multiple molecules.
The term "compound" as used herein, or a recitation of a specific compound (such as 5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin)is used to denote a molecular moiety of unique, identifiable chemical structure. A molecular moiety ("compound") may exist in a free species form, in which it is not associated with other molecules. A compound may also exist as part of a larger aggregate, in which it is associated with other molecule(s), but nevertheless retains its chemical identity. A solvate, in which the molecular moiety of defined chemical structure ("compound") is associated with a molecule(s) of a solvent, is an example of such an associated form. A hydrate is a solvate in which the associated solvent is water. The recitation of a "compound" refers to the molecular moiety itself (of the recited structure), regardless whether it exists in a free form or and an associated forms.

The term "5(S)-(2’-hydroxyethoxy)-20(S)-camptothecin" is intended to cover not only the more active lactone form, but also the less active carboxylate form, including any salts formed therefrom.
The term "inhibit" and variations thereof refer to any reduction of a cellular activity, such as topoisomerase I activity, which can be measured via methods described herein.
The term "pharmaceutically acceptable" as used herein refers to those active agents, hydrates, solvates, salts, polymorphs, carriers, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.
The term "individual", "subject" and "patient" as used herein refer to any animal for whom diagnosis, treatment, or therapy is desired. The term "animals" as used herein refers to humans and other mammals, as well as other animals.
The terms "treating" and "treatment" of a state, disorder, disease or condition as used herein refer to (1) preventing or delaying the appearance of clinical symptoms of the state, disorder, disease or condition developing in a patient that may be afflicted with or predisposed to the state, disorder, disease or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder, disease or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to a patient to be treated is either statistically significant or at least perceptible to the patient and/or to the physician.
The terms "effective amount" and "therapeutically effective amount" as used herein refer to the amount of a compound that,

when administered to a patient for treating a state, disorder, disease or condition, is sufficient to effect such treatment. The effective amount or therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, physical condition and responsiveness of the individual to be treated.
The terms "delivering" and "administering" as used herein refer to providing a therapeutically effective amount of an active agent to a particular location or locations within a patient causing a therapeutically effective concentration of the active ingredient at the particular location or locations. This can be accomplished, e.g., by local or by systemic administration of the active ingredient to the host.
The term "composition" as used herein refers to a product comprising the specified agent or agents, as well as any product, which results, directly or indirectly, from combination of the specified ingredients. A "pharmaceutical composition" is intended to include the combination of an active agent or agents with one or more pharmaceutically acceptable carriers, inert or active, making the composition suitable for diagnostic or therapeutic use in vivo, in vitro or ex vivo. The compositions can also include stabilizers, preservatives, adjuvants, fillers, flavors and other excipients.
The term "substantially free from" is defined hereinbelow.
Example 26 of the M39 patent described the preparation of 5-(2'-hydroxyethoxy)-20(S)-campthothecin (hereinafter "5(RS)-CPT"), which is a mixture of R- and S-diastereomers, namely, 5 (R) - (2 ' -hydroxyethoxy)-20(S)-campthothecin (hereinafter, "5(R)-CPT" and 5(S)-(2'-hydroxyethoxy)-20(S)-campthothecin (hereinafter "5 (S)-CPT") respectively. It has now been discovered that 5 (S)-CPT has unexpectedly superior properties in comparison to 5(R)-CPT and 5(RS)-CPT. The inventors have found that 5(S)-CPT differs from the diastereomeric mixture 5(RS)-CPT in several important characteristics, including, for example, toxicity, in vivo anti-tumor activity, bioavailability, X-ray powder diffraction pattern,

thermal stability, and topoisomerase I inhibition. Furthermore, it has now been also discovered that 5 (S) -CPT substantially free from 5(R)-CPT is a desirable anti-cancer pharmaceutical compound.
The activity of the Topoisomerase I enzyme is associated with cytotoxic activity. Thus, the inhibitory effect on topoisomerise I activity is predictive of anti-cancer efficacy for CPT compounds (Curr Pharm Des. 2002, 8(27), 2505-20; Clin Cancer Res. 2002, (3), 641-61/ Ann N Y Acad Sci. 2000, 922, 11-26) In vitro activities of 5 (S)-CPT and 5(R)-CPT, which were substantially free of one another, in inhibiting topoisomerise I enzyme had been measured and are set forth in TABLE I (the assay is provided in EXAMPLE 3):
TABLE I

With reference to TABLE I, it can be observed that 5(S)-CPT is about 21 folds more active than 5 (R) -CPT and about 12 times more active than 5(RS)-CPT. The inventors have recognized that such differences in activity would not be expected based on structural differences between the diastereomers since it is known that, particularly in view of the importance of the E-ring in enzyme activity.
The inventors have further discovered that 5 (S)-CPT demonstrates better in vivo activity against several tumor xenograf ts in nude mice versus 5 (RS) -CPT. The results of the in vivo comparison studies between 5(S)-CPT and 5(RS)-CPT in nude mice against NCI-H460 (human small cell lung carcinoma) xenografts are presented in TABLE 2 (please see EXAMPLE 4 for procedure and methodologies). TABLE 2

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As shown in TABLE II, the administration of 5 (S) -CPT led to unexpectedly superior inhibition of tumor growth in comparison with the administration of 5(RS)-CPT at identical doses (68% vs 6 0% at 2 mg/kg, and 76% vs 64% at 4 mg/kg) without increase in mortality.
Likewise, 5(S)-CPT showed unexpectedly superior in vivo activity, in comparison with 5(RS)-CPT in several osteosarcoma tumor models. In vivo comparison studies between 5(S)-CPT and 5(RS)-CPT in nude mice against osteosarcoma models are presented below.
All mice bearing sc tumors measuring approximately 0.2 - 1 cm in diameter were treated with test compound by oral gavage using [ (dx5)2]3 schedule. Tumor diameters were measured every 7 days using Vernier calipers and tumor volumes were calculated, assuming tumors to be spherical, using the formula [n/S) x d3] , where d is the mean diameter. The tumor response to the test compound was defined as follows. For individual tumors, PR was defined as a volume regression >50%, but with measurable tumor at all times. CR was defined as disappearance of measurable tumor mass at some point within 12 weeks after initiation of therapy. Maintained CR is defined as no tumor re-growth within a 12-week study time frame. This time point was chosen because all studies lasted at least 12 weeks. Mice that died before the end of the 12-week study time, and prior to achieving a response, were considered as failures for tumor response. The results (dose of 28 mg/kg) are presented in TABLE 3: TABLE 3

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6+ : Complete Regression Maintained
5+ : Complete Regression
4+ : Partial Regression
3+ : Stable Disease
As seen with reference to TABLE 4, administration of 5(S)-CPT led to unexpectedly superior results in comparison with the administration of 5(RS)-CPT, as indicated by complete regression (6+) achieved with 5(S)-CPT in all four xenograft lines.
The above data have convinced the inventors that 5(S)-CPT has unexpectedly superior activity/potency profile in several test models. Furthermore, the inventors have also recognized that while 5(S)-CPT is substantially more potent than 5(R)-CPT, the increase in potency is unexpectedly unaccompanied by a corresponding increase in toxicity.
TABLE 4 below shows results of an in vitro cell proliferation comparison study between 5(s)-CPT and 5(R)-CPT (the protocol is appended in EXAMPLE 5. Cell proliferation was evaluated by Sulphorhodamine B (SRB) assay where the amount of dye bound to the cells after staining gives a measure of cell growth. See JNCI, vol 83, No. 11, June 5, 1991, which is incorporated herein by reference.
TABLE 4

The in vitro potency of the diastereomers against cancer cell lines had been compared with their in vitro toxicity in healthy cells. TABLE 5 below presents the results of the bone marrow toxicity comparison study in human cells (for assay, please see EXAMPLE 6):
TABLE 5

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With reference to TABLES 4 and 5, it can be seen that while 5(S)-CPT is unexpectedly almost 3 times more potent in this assay than 5(R)-CPT, the toxicities of both diastereomers are comparable. In fact, if the safety margin is estimated as the ratio of GI90 for human cell toxicity to GI 50 for anticancer activity. Accordingly, the estimated safety margins are set forth in the below TABLE 6:

With reference to TABLE 6, the inventors recognized that 5(S)-CPT is unexpectedly showing far better safety profile than 5(R)-CPT.
The sum of the results set forth above have indicated to the inventors that 5(S)-CPT is unexpectedly superior to 5(R)-CPT and 5(RS)-CPT as a pharmaceutical compound for treatment of cancer. In other words, the inventors have recognized that 5(S)-CPT is the substance of choice with respect to treatment of cancer in comparison with the R-diastereomer and the mixture of diastereomers. In fact, further work has also demonstrated to the inventors that it is unexpectedly important to minimize the amount of the 5(R)-CPT present in the 5 (S)-CPT to be given to patients.
In one study, 5(S)-CPT (2.5 mg/kg) and 5(RS)-CPT (5 mg/kg, including 2.5 mg/kg of 5 (S)-CPT in the mixture) have been administered to male Wistar Rats to evaluate oral pharmacokinetics. The results of the study are presented in TABLE 7A. TABLE 7A

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In another study, 5 (S)-CPT (2.5 mg/kg) and 5(RS)-CPT (5 mg/kg, including 2.5 mg/kg of 5(S)-CPT in the mixture) have been administered to male Swiss Albino Mice to evaluate oral pharmacokinetics. The results of the study are presented in TABLE 7B. TABLE 7B

With reference to TABLES 7A and 7B, the "Contribution of 5(S)-CPT" is the Area Under Curve that can be attributed to the S-diastereomer in the RS diastereomeric mixture. As can be seen from TABLES 7A and 7B, presence of 5 (R) -CPT substantially and unexpectedly decreases bioavailability of the desired 5 (S) diastereomer. Moreover, the inventors have reasons to believe that such unexpected decrease in bioavailability for the desired diastereomers will also be observed in human patients. On the basis of the above, the inventors have recognized that minimization of the amount of the R diastereomers impurity in 5(S) CPT is highly desirable.
Thus, there is provided 5(S) -(2'-hydroxyethoxy)-20(S)-campthothecin of the structure:

which is substantially free from 5(R)-(2'-hydroxyethoxy)-20(S)-campthothecin. The term "substantially free from 5(R)-(2'-hydroxyethoxy)-20(S)-campthothecin" denotes 5(S)-CPT containing less than 2% of 5 (R) -CPT as an impurity. In particular
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embodiments, there is provided 5 (S)-(2'-hydroxyethoxy)-20 (S)-campthothecin containing less than 1.5% of 5 (R) - (2’-hydroxyethoxy) -20 (S) -camptothecin w/w, or less than about 1.0% 5 (R) - (2 ' -hydroxyethoxy) -20 (S) -camptothecin w/w, or less than about 0.5% 5 (R) - (2 ' -hydroxyethoxy) -20 (S) -camptothecin w/w, or less than about 0.1% 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin w/w. FIGS. 1A and IB provide HPLC chromatograms that illustrate purity of non-limiting examples of 5(S) CPT according to the invention.
In one embodiment, 5 (S)-(2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5 (R)-(2’-hydroxyethoxy)-20(S)-camptothecin is isolated in crystalline form, which exists substantially free from water (i.e., essentially anhydrous). The term "substantially free from water" means the presence of less than about 1.0% water w/w and even more preferably less than about 0.5% water w/w as measured by Karl Fisher analysis or TGA. In this form, 5 (S)- (2'-hydroxyethoxy)-20(S)-camptothecin has the advantageous property that it is non-hygroscopic at storage conditions of up to 40° C ± 2° C/65% RH ± 5% RH for 6 months as measured by Karl Fisher analysis. The product is stable in 6 months accelerated conditions (40° C ± 2° C/65% RH ± 5% RH) and stable for six months at 30° C ± 2° C/65% ± 5% RH
An example of an X-ray powder diffraction pattern of crystalline 5(S)-CPT substantially free from 5(R)-CPT is shown in FIG. 2. For comparison, the X-ray powder diffraction patterns for 5(RS) CPT, produced according to U.S. Patent No. 6,177,439, and 5 (R) CPT are shown in FIGS. 3A and 3B, respectively. Several substantial peak differences are noted, such as, for example, at about 7.4, 9.0, 11.4 and 13.0 degrees 29, indicating different solid state structures. X-ray powder diffraction spectra were determined using a Rigaku D/Maz 2200 diffractometer equipped with horizontal goniometer in 9/20 geometry. The X-ray tube used was a Cu K-alpha with a wavelength of 1.5418 A at 50 KV and 34 mA. The divergence and scattering slits were set at 0.5° and the receiving slit set at 0.15 mm. Diffracted radiation was detected by
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scintillation counter detection, 9 to 29 continuous scan at 3 degrees/minute from 3 to 45 degrees.
Crystalline 5 (S)- (2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R)- (2'-hydroxyethoxy)-20(S) -camptothecin may also be characterized by a single melting endotherm peak at about 23 7° C as measured by DSC and shown in FIG. 4. In contrast, the DSC profile for 5(2'-hydroxyethoxy)-20(S)-camptothecin as produced according to U.S. Patent No. 6,177,439 and shown in FIG. 5A is much more complex, and shows an endotherm peak at about 39° C, a second endotherm peak at about 173° C and a third endotherm peak at about 2 07° C, consistent with the 190° C melting point listed in U.S. Patent No. 6,177,439 for this compound. And DSC profile for 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin has shown in FIG. 5B shows an endotherm peak at about 63° C, a second endotherm peak at about 186° C and a third endotherm peak at about 205° C. This again confirms the different solid state structures of the two compounds. Further, the higher melting point indicates a greater stability of 5 (S)- (2’-hydroxyethoxy)-20(S)-camptothecin. DSC was performed using a Shimadzu DSC-50 calorimeter. Samples were placed in an aluminum pan, the weight accurately recorded and the pan covered with a lid and left undamped. Each sample was equilibrated and heated at a rate of 10° C/minute under nitrogen atmosphere.
5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin may be obtained by the following processes, which form a further aspect of the present invention.
5-(2'-hydroxyethoxy)-20(S)-camptothecin, obtained by the process disclosed in U.S. patent No. 6,177,439 or a similar process, is suspended in a suitable organic solvent, such as methanol, ethanol, propanol, isopropanol, n-butanol or THF, and ref luxed over a period of about 2-3 hours. The suspension is slowly cooled to about 40-45° C such that a complete precipitate is formed. The precipitate is isolated using conventional techniques, such as filtration, and can be washed with the same or
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other suitable organic solvent and dried under vacuum to yield solid 5(S)-(2’-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R) -(2'-hydroxyethoxy)-20(S)-camptothecin. The product can be further enriched by repeatedly refluxing from about 2-4 times in the same or other suitable organic solvent.
5 (S)-CPT as described herein is intended as an agent for treatment of cancer. Preferred route of administration is oral. 5(S)-CPT may be administered to a patient in the form of a pharmaceutical composition. In addition to the active compound, the pharmaceutical composition will generally contain one or more pharmaceutically acceptable carriers comprising excipients, coatings, auxiliaries and the like known in the art. Accordingly, another aspect of the present invention is directed to a pharmaceutical composition comprising 5(S)-(2'-hydroxyethoxy) -20(S)-camptothecin substantially free from 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin and one or more pharmaceutically acceptable carriers. The form of the composition will depend in part upon the desired route of administration and include, for example, pills, tablets, capsules, and the like. Formulations for oral are preferred. The pharmaceutical composition of the present invention can be formed by simple admixture between the active compound and the pharmaceutically acceptable carrier(s) using methods well known in the art. Examples of pharmaceutically acceptable carriers and methods of manufacture for various pharmaceutical compositions may be found in, e.g., A. Gennaro (ed.), Remington1s Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co. , Easton, Pa. The pharmaceutical composition typically contains from 0.1 to 50% w/w, preferably 1 to 20% w/w, of active compound, the remainder of the composition being the pharmaceutically acceptable carrier or carriers, diluents or solvents. Preferably, the pharmaceutical composition is in a unit dosage form containing appropriate quantities of the active compound, e.g., an amount effective to achieve the desired purpose.
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For any compound, the therapeutically effective dose can be estimated initially either in cell culture or in animal models. The therapeutically effective dose refers to the amount of active compound that ameliorates the condition or its symptoms. Therapeutic efficacy and toxicity in cell cultures or animal models may be determined by standard pharmaceutical procedures (e.g., ED50: the dose therapeutically effective in 50% of the population; LD50: the dose lethal to 50% of the population). The data obtained from cell culture and animal models can then be used to formulate a range of dosage for the compound for use in patients. A typical recommended daily dosage regimen will generally range from about 0.001 mg/kg/day to about 200 mg/kg/day, preferably from about 0.01 mg/kg/day to about 100 mg/kg/day, preferably from about 0.1 mg/kg/day to about 10 mg/kg/day, preferably from about 0.1 mg/kg/day to about 5 mg/kg/day.
The actual dosage employed and the frequency of administration may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required.
Specific embodiments according to the methods of the present invention will now be described in the following examples. The examples are illustrative only, and are not intended to limit the remainder of the disclosure in any way.
EXAMPLE 1:
Preparation of 5 (S)-(2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R)-(2-hydroxyethoxy)-20(S) -camptothecin.
5-(2'-hydroxyethoxy)-20(S)-camptothecin (75 grams) (prepared as described in U.S. Patent No. 6,177,439) was suspended in n-butanol (600 ml) and ref luxed over a period of 2-3 hours. The reaction mass temperature was slowly lowered to 40-45° C, filtered, washed with n-butanol (15-20 ml) and dried under vacuum
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at 50-55° C to yield solid 5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R)-(2-hydroxyethoxy)-20 (S)-camptothecin. The product was further enriched by repeatedly refluxing in n-butanol (generally 2-4 times; yield 25-35 grams). The final dried product was characterized by the X-ray powder diffraction pattern shown in FIG. 1, with specific peaks at about 4.68, 7.22, 9.4, 11.02, 12.00, 14.14, 14.54, 15.20, 16.22, 17.10, 17.32, 17.94, 18.92, 19.16, 19.70, 21.86, 22.22, 22.74, 23.28, 23.72, 24.22, 24.4, 25.36, 26.42, 27.12, 27.66, 28.58, 29.12, 29.46, 30.62, 30.78, 31.42, 31.94, 32.46, 32.78, 33.30, 33.54, 33.98, 35.18, 36.74, 37.62, 38.28, 38.92, 39.82, 40.38, 42.08, 42.22 and 43.98 degrees 29. EXAMPLE 2:
Preparation of 5 (S)-(2’-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R)-(2’-hydroxyethoxy)-20(S)-camptothecin.
5-(2'-hydroxyethoxy)-20(S)-camptothecin (75 grams) (prepared as described in U.S. Patent No. 6,177,43 9) was suspended in THF (1125 ml) and refluxed over a period of 2-3 hours. The reaction mass temperature was slowly lowered to 40-45° C, filtered, washed with n-butanol (15-20 ml) and dried under vacuum at 50-55° C to yield solid 5(S)-(2'-hydroxyethoxy)-20(S)-camptothecin substantially free from 5(R)-(2'-hydroxyethoxy)-20(S)-camptothecin. The product was further enriched by repeatedly refluxing in n-butanol (generally 2-4 times; yield 15-20 grams). The final dried product was characterized by the X-ray powder diffraction pattern shown in FIG. 1. EXAMPLE 3:
Topoisomerase I Assay:
Topoisomerase I introduces transient nicks in DNA at specific sites. Detection of these transient DNA nicks requires trapping the enzyme on DNA in a nicked intermediate complex using protein denaturants. The resulting covalent DNA/topo I complexes contain nicked open circular DNA which can be detected by agarose gel electrophoresis (with ethidium bromide). Trapping nicked
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intermediates is relatively inefficient; however, inhibitors, such as the natural product camptothecin, stabilize the intermediate and lead to an increase in the nicked DNA product. This forms the basis for a mechanistic drug screen designed to allow detection of agents that affect topoisomerase I by stabilizing the cleaved intermediate complex. The TopoGEN Topo I Drug Screening Kit is designed to allow the investigator to quickly identify novel inhibitors of topoisomerase I. The kit will allow detection of novel compounds that either stabilize the nicked intermediate or otherwise inhibit catalytic activity of topoisomerase I.
Assay KIT used: Topogen Drug screening kit,
Manufacturer: TOPOGEN, Cat No: 1018 .
Each reaction mix contains:
a. lOx Reaction buffer : 2 ul
b. TOPO I enzyme : 2 ul
c. pHOT I DNA : 1.2 ul (0.5ug)
d. Water : 14.8 ul
Total : 20 ul
Protocol
The above reaction mixture is incubated at 37°C for 3 0 minutes. Reaction is terminated by adding 2 ul of 10% SDS and the mixuture is vortexed rapidly (SDS should be added while at 3 7°C as cooling the tubes might reseal the nicked DNA) . 10X Dye about 2.5 ul per tube is added and equal volumes of mixture of Chloroform and Isoamyl alcohol (24:1) is added and centrifuged at 13000 rpm for 10 minutes. Samples are loaded on a 1% agarose gel and electrophoresed for 1 hour at 80 volts. Gel was then viewed on UV transilluminator and then the densitometric estimation of the bands was done.
Calculations:
The density of the DNA bands of both super coiled and relaxed forms of DNA was measured using densitometer. The band intensity of treated (with single cone, of drug) and without drug ie: Control were recorded. The percentage of relaxed form compared to
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the supercoiled was calculated for all the lanes including treated and control.
% inhibition of Topoisomerase activity = (100- (100 X (1/ % inhibition in Control) X % inhibition in treated)) EXAMPLE 4:
Protocol of comparision study on 5(RS)-CPT AND 5(S)-CPT against NCI-H460 xenograft in nude mice
To perform the NCI-H460 xenograft study, NCI-H460 tumor pieces measuring -60 mm3 were implanted in the space of dorsal lateral flanks of female athymic nude mice to initiate tumor growth. When the tumors were grown to -150-1000 mm3, animals were randomized into groups of five prior to initiating therapy. Each gram of 5-(2’-hydroxyethoxy)-20(S)-camptothecin was formulated to contain 102.65 mg active compound, 801.62 mg hydroxylpropyl beta cyclodextran, 80.62 mg dextrose anhydrous and 13.33 mg sodium carbonate. Each gram of 5 (S)-(2'-hydroxyethoxy)-2 0(S) -camptothecin was formulated to contain 105.57 mg active compound, 800.99 mg hydroxylpropyl beta cyclodextran, 80.13 mg dextrose anhydrous and 13.34 mg sodium carbonate. Each gram of placebo was formulated to contain 895.2 mg hydroxylpropyl beta cyclodextran, 89.52 mg dextrose anhydrous and 14.9 mg sodium carbonate. Each formulation was dissolved in 2 ml sterile water and administered through oral route in a (dx5)2 schedule. Tumor diameters were measured twice a week using a vernier caliper.
Tumor volumes were calculated assuming tumors to be ellipsoid using the formula:
V = (Dxd2) /2,
where V (mm3) is tumor volume, D is longest diameter in mm and d is shortest diameter in mm. Change in tumor volumes (A) for each treated (T) and control (C) group were calculated by subtracting the mean tumor volume on the first day of treatment (starting day) from the mean tumor volume on the specified observation day. These values were used to calculate a percentage growth (%T/C) using the formulas:
%T/C = (AT/AC) x 100, where AT > 0, or
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%T/C = (rT/rTi) x 100, where AT < 0 and
Ti is the mean tumor volume.
Percentage tumor growth inhibition (%TGI) was then calculated using the formula:
%TGI = 100 - %TC.
All the mice bearing subcutaneous tumors measuring approximately 150 - 800mm3 were treated with test compound through oral gavage using a (dx5)2 schedule. Tumor diameters were measured twice in a week using vernier calipers and tumor volumes were calculated assuming tumors to be ellipsoid using the formula V = (Dxd2)/2 where V (mm3) is tumor volume, D is longest diameter in mm., and d is shortest diameter in mm. Changes in tumor Volumes (r volumes) for each treated (T) and control (C) group are calculated, by subtracting the mean tumor volume on the first day of treatment (starting day) from the mean tumor volume of on the specified observation day. These values are used to calculate a percentage growth (%T/C) using the formula
%T/C = (rT/rC) X 100 where rT > 0 or =(rT/rTi) X 100 where AT < 0 , where Ti is the mean tumor volume at the start of treatment. Percentage tumor growth inhibition was calculated using the formula, Percentage Tumor growth inhibition = 100 - %T/C.
Tumor regressions are defined as partial if the tumor volume decreases to 50% or less of the tumor volume at the start of the treatment without dropping below to 63mm 3. Complete regression is defined if the tumor volume drops to below measurable limits (< 63mm 3)
The percentage body weight change in comparison to starting day body weight of each animal is calculated using the formula ,Percentage Body weight change = [(Body weight on specified observation day - Body weight on starting day)/ Body weight on starting day] X 100. The other parameter observed was mortality. EXAMPLE 5: Protocol for In Vitro Cell growth assay.
Briefly, cells were seeded on a 96-well cell culture plates at a concentration of 10000 cells per well and incubated at 37°C in
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C02 incubator. Twenty-four hours later cells were treated with different concentrations of andrographolide dissolved in DMSO to a final concentration of 0.05% in the culture medium and exposed for 48 h. Cells were fixed by adding ice-cold 50% trichloroacetic acid (TCA) and incubating for 1 h at 4°C. The plates were washed with distilled water, air dried and stained with SRB solution (0.4% wt/vol in 1% Acetic acid) for 10 min at room temperature. Unbound SRB was removed by washing thoroughly with 1% acetic acid and the plates were air-dried. The bound SRB stain was solubilized with lOmM Tris buffer, and the optical densities were read on a spectrophotometric plate reader at a single wavelength of 515 nm. At the time of drug addition separate reference plate for cell growth at time 0 h (the time at which drugs were added) was also terminated as described above. From the optical densities the percentage growths were calculated using the following formulae, If T is greater than or equal to TO, percentage growth = 100 x [ (T-To) / (C-To) ] and if T is less than To, percentage growth = 100 x [ (T-To)/To)] , Where T is optical density of test, C is the optical density of control and To is the optical density at time zero. From the percentage growths a dose response curve was generated and GI50 values were interpolated from the growth curves.
EXAMPLE 6: Protocol for Human Bone Marrow Assay:
Methocult™ GF (Cat No: H4 534, Poietics, Biowhittakar, USA) medium comprising Methycellulose in Iscove's MDM, Fetal bovine serum, Bovine serum albumin, 2-Mercaptoethanol, L-Glutamine, rhStem cell factor, rhGM-CSF and rhIL-3 was used for the assay. Human bone marrow mononuclear cells (Cat NO.2M-125C, Poietics, Biowhittakar, USA) were mixed with Methocult GF and the cell
density was adjusted to 3 x 105 cells/ml. From this 500|aL aliquots were made and 2. 5|iL of 20X drug solution or vehicle was added the each aliquot and mixed thoroughly. 100|aL of clonogenic medium was plated into each well and the plates were allowed to gel at 4°C for 15 minutes. Plates were incubated at 37°C in a fully
humidified atmosphere of 5% C02 incubator for 14 days. CFU-GM
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colonies were counted under an inverted microscope as aggregates of 50 cells or more. The percentage colony inhibition was calculated using the following formula:
100-[ (number of colonies in drug treated wells/Number of colonies in control wells) X 100].
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.
All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.
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