FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rale 13)
TITLE OF THE INVENTION «AN IMPROVED COMPOSITION OF GEMCITABINE"
We, BA Research India Limited, of BA Research House, Opposite "Pushparaj Towers", Nr. Judges Bunglows, Bodakdev, Ahmedabad-380054, Gujarat, India.
The following specification particularly describes the nature of the invention and the manner in which it is performed:

FIELD OF THE INVENTION
The present invention relates to the composition of anti-tumor chemotherapeutics and more particularly to delivery of the anti-tumor chemotherapeutic, Gemcitabine. BACKGROUND
Cancer is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and the ability to invade local tissues and metastasize. These neoplastic malignancies affect, with various degrees of prevalence, every tissue and organ in the body. A multitude of therapeutic agents have been developed over the past few decades for the treatment of various types of cancer. The most commonly used types of anticancer agents include: DNA-alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, a folate antagonist, and 5-fluorouracil, a pyrimidine antagonist), microtubule disrupters (e.g., vincristine, vinblastine, paclitoxel), DNA intercalators (e.g., doxorubicin, daunomycin), and hormone therapy (e.g., tamoxifen, flutamide). The ideal antineoplastic drug would kill cancer cells selectively, with a wide therapeutic index relative to its toxicity towards non-malignant cells. It would also retain its efficacy against malignant cells, even after prolonged exposure to the drug. Unfortunately, none of the current chemotherapies possess an ideal profile. Most possess very narrow therapeutic indexes and, in practically every instance, cancerous cells exposed to slightly sublethal concentrations of a chemotherapeutic agent will develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents.
The development of new anticancer agents has given rise to new treatment regimens and new combinations that are proving more effective in combating this disease. U.S. Pat. No. 5,415,869, which is incorporated by reference, discloses paclitoxel tumor-active analogs solubilized using one or more negatively charged phospholipids and one or more zwitterionic phospholipids. The phospholipid mixture entraps paclitoxel or the analog in a liposome. The liposome is in the form of particles having a size of 0.025 to 10 microns, with substantially no crystals of paclitoxel or the analog.
U.S. Pat. No. 5,580,575, which is incorporated by reference, discloses a therapeutic drug delivery system comprising gas-filled microspheres and a therapeutic drug, as well as, methods for employing such microspheres in therapeutic drug delivery. The preferred microspheres of the disclosure are gas-filled liposomes with an encapsulated drug. Methods of preparing such liposomes in drug delivery applications are also disclosed. WO 99/13914, incorporated herein by reference, discloses that paclitoxel, and other slightly water soluble drugs can be formulated without CREMOPHOR.RTM.EL or other toxic solubilizers by forming a water soluble homogeneous complex with plasma proteins, such as

human serum albumin (HSA) or human gamma globulin (.gamma.-globulin). As disclosed by WO 99/13914 homogeneous aqueous solutions up to at least 4.68 mM paclitoxel (4 mg/mL) can be formulated using HSA. The plasma proteins act as a slow release depot of paclitoxel. WO 99/13914 further discloses a dosage range of paclitoxel -HSA complex containing 70-280 mg of paclitoxel per treatment. Such formulations can be made bio-equivalent to the conventional CREMOPHOR.RTM.EL containing formulations
Other formulations for administering paclitoxel are disclosed in U.S. Pat. Nos. 5,504,102 and 5,407,683, incorporated herein by reference.
In addition, the slow infusion of CREMOPHOR.RTM.EL solutions has been studied as a means of avoiding or ameliorating the side effects of the CREMOPHOR.RTM.EL vehicle. The most common dosage is 135-175 mg/m. CREMOPHOR.RTM.EL, which is administered over a 3 hour, 6 hour, or 24 hour dosage schedule. (See U.S. 5,641,803, and 5,621,001, both incorporated herein by reference.) Other dosing schedules have been suggested to reduce toxic side effects, including 96 hour infusion every 21 days (U.S. Pat. No. 5,496,846, incorporated herein by reference) and 60-180 minutes, repeated a plurality of times during a 21 day period, each infusion separated by an interval of between 4 to 5 days.. The limitations of current chemotherapy reservoir technology is probably due to the retention of the chemotherapeutic drug only on the tumor periphery or at the injection site due to the poor penetration and distribution of the drug as a result of the neoplasm's high interstitial fluid pressure. A more potent anti-tumor effect can be achieved by targeting the chemotherapy directly to the tumor, i.e., intratumorally, rather than by systemic infusion. We now report a method of delivering an anti-cancer chemotherapeutic, such as Gemcitabinel, by first administered Gemcitabinel by intratumoral injection and thereafter administering Gemcitabinel by intravenous injection. This invention takes advantage of the lower toxicity and side effects of Gemcitabinel/plasma solutions, and the ability of plasma proteins, such as HSA, to act as a slow release depot for Gemcitabinel.
Accordingly, it is one object of the present invention to provide compositions which directly or indirectly are toxic to actively dividing cells and are useful in the treatment of cancer.
A further object of the present invention is to provide methods for killing actively proliferating cells, such as cancerous, bacterial, or epithelial cells, and treating all types of cancers, and generally proliferative conditions. A further object is to provide methods for treating other medical conditions characterized by the presence of rapidly proliferating cells, such as psoriasis and other skin disorders.

SUMMARY OF THE INVENTION
The present invention provides for a method of delivering Gemcitabinel, the method comprising an intratumoral dose of a Gemcitabinel formulation and an intravenous infusion of Gemcitabinel wherein the intravenous infusion occurs about 24 hours to about 7 days after the intratumoral dose. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for a method of delivering a Gemcitabinel. According to the invention Gemcitabinel, as a Gemcitabinel formulation, is first brought into contact with substantially all the cells of a solid tumor, by an intratumoral dose. Thereafter Gemcitabinel is administered by intravenous infusion. The Gemcitabinel administered by intravenous infusion may be the same Gemcitabinel formulation used in the intratumoral dose. Alternatively, the Gemcitabinel may be administered by infusion of Gemcitabinel in any other soluble form.
While not being bound by theory, it is believed that the intratumoral dose of the Gemcitabinel formulation induces apoptosis within the tumor by slowly releasing Gemcitabinel into the tumor over a period of twenty-four hours to one week. The cell death that occurs within the tumor results in the collapses of the tumor structure. The collapsed tumor allows access of the second intravenous dose of Gemcitabinel to reach inside the partially collapsed tumor structure. One of skill in the art will recognize that, the invention is not limited to methods which function in this manner. Intratumoral Dose of Gemcitabinel Formulation
One aspect of the present invention provides for introducing a Gemcitabinel formulation intratumorally. For example, in one embodiment of the present invention, the intratumoral dose of Gemcitabinel formulation may be injected intratumorally using a syringe pump. The flow rate and pressure of the syringe pump will depend upon the tumor to be treated. The flow rate of the syringe pump may vary from about 0.0167 ml/min to about 0.5 ml/min. The preferred flow rate will deliver the Gemcitabinel formulation to greater than 90% of the tumor volume while delivering essentially no Gemcitabinel outside the tumor.
The Gemcitabinel formulation is preferably a soluble form of Gemcitabinel comprising a Gemcitabinel/plasma protein complex. As used herein, Gemcitabinel/plasma protein complex refers to Gemcitabinel in a water-ethanol solution containing a solubilizing amount of plasma protein wherein the Gemcitabinel forms a non-covalent complex with the plasma protein. Preferably the plasma protein is HSA or .gamma.-globulin. Most preferably the plasma protein is HSA. One of skill in the art will understand that Gemcitabinel/plasma protein is not limited to the use of these two proteins and includes any plasma protein capable

of forming a non-covalent Gemcitabinel/plasma protein complex and thereby solubilizing Gemcitabinel.
While not being bound by theory, it is proposed that administering a soluble form of Gemcitabinel, such as a Gemcitabinel/plasma protein complex, increases drug efficacy by promoting Gemcitabinel diffusion. Increased diffusion promotes apoptosis tumor cell death not only in the immediate zone of the injection but also at sites further into the tumor where the Gemcitabinel has migrated.
The mass of Gemcitabinel formulation delivered intratumorally depends upon the size of the tumor, and can range up to about 280 mg of Gemcitabinel. Preferably, the intratumoral mass of Gemcitabinel is from about 1 to about 60 mg of Gemcitabinel.
The volume of the dose is preferably about 1/4 to about 1/12 the tumor volume. Most preferably the volume of the dose is about 1/10 of the tumor volume. The preferred concentration of the Gemcitabinel formulation is about 4 to about 10 mg/ml of Gemcitabinel, or about 3.4 to about 8.5 mM Gemcitabinel.
Thus, a tumor with a 4 cm diameter has a volume of 33 cc. Consequently, a 6 ml of a 10 mg/ml dose of Gemcitabinel liquid delivered into the tumor results in a dose of 60 mg Gemcitabinel which is approximately the maximal intratumoral injection dose. If the initial intratumoral administration of the Gemcitabinel formulation does not substantially shrink the solid tumor, an additional intratumoral dose of the Gemcitabinel formulation may be administered. The additional intratumoral dose may be at an identical, at a greater, or at a lower concentration of the Gemcitabinel formulation then the initial intratumoral dose, in one embodiment of the present invention, the Gemcitabinel/plasma protein may be administered by multiple intratumoral injections within a short period of time. The invention provides for multiple intratumoral injections of the Gemcitabinel formulation administered within 24 hours.
While not being bound by theory, administering the Gemcitabinel formulation by frequent intratumoral injections may increase the efficacy of Gemcitabinel by inducing apoptosis at various stages of the cell cycle. Intravenous Infusion of Gemcitabinel
Methods of delivering an anti-tumor chemotherapeutic by intravenous infusion are well known in the art and are described for example in U.S. Pat. Nos. 5,696,153, 5,496,846, and 5,641,803.
In one embodiment of the present invention, the Gemcitabinel is administered by intravenous infusion about twenty-four hours to about 1 week following the intratumoral dose as a continuous infusion. The intravenous dose is typically administered over about 3 to about
5

12 hours. The Gemcitabinel administered by intravenous infusion may be the Gemcitabinel formulation used in the intratumoral dose administered as a saline solution of 5% dextrose or normal saline. Alternatively, the Gemcitabinel may be administered by infusion of Gemcitabinel in any other soluble form.
When the Gemcitabinel administered by intravenous infusion comprises the Gemcitabinel/plasma protein complex, the intratumoral or the intravenous treatment may be repeated in cycles. The administration of the Gemcitabinel/plasma protein complex may be repeated because of the decreased hypersensitivity reaction from the Gemcitabinel Gemcitabinel/plasma protein complex compared to TAXOL.RTM
In one embodiment, the intravenous infusion of Gemcitabinel comprises administering a plurality of repeated intravenous infusions subsequent to the intratumoral dose, wherein each infusion is separated by about seven days.
Another embodiment comprises administering an additional intravenous infusion of the Gemcitabinel formulation, about 4 to about 21 days subsequent to the intravenous infusion.
Another embodiment comprises an additional intratumoral dose administered subsequent to the intravenous dose. The additional intratumoral dose is preferably administered about 4 to about 21 days subsequent to the intravenous infusion.
In an alternative embodiment of the invention, the intravenous dose may be administered by sotubilizing Gemcitabinel in CREMOPHOR.RTM.EL ethanol solutions. The solution of this embodiment comprises 6 mg/mL Gemcitabinel, corresponding to a Gemcitabinel concentration of about 7 mM, which is diluted prior to infusion with 0.9% sodium chloride injection, U.S.P, 5% dextrose injection, U.S.P, 5% dextrose and 0.9% sodium chloride injection, U.S.P, or 5% dextrose in Ringer's injection to a final concentration of 0.3 to 1.2 mg/mL. The maximum TAXOL.RTM. concentration which can be administered by intravenous infusion using this formulation is about 0.6 mg/mL.
The intravenous dose is preferably in the range of about 100 to about 200 mg/m.sup.2. More preferably the intravenous dose is in the range of about 135 to about 175 mg/m.sup.2.
The mass of Gemcitabinel administered by intravenous infusion is preferably between about about 70 to about 280 mg.
The function and advantage of these and other embodiments of the present invention will be more fully understood from the examples below. The following examples are intended to illustrate the benefits of the present invention, but do not exemplify the full scope of the invention.

EXAMPLE 1
Improved Spread of Evan's Blue-Albumin in a Human Mammary Adenocarcinoma MCF7 Xenograft in Immunocompromised Mice when Injecting Intratumorally Under Pressure as a Model for Gemcitabinel/HSA Spread in the Tumor. Purpose
The purpose of the study is to assess the efficiency of spread of a solution of Evan's blue dye--albumin complex in a tumor when injected intratumorally at different flow rates. The complex of the dye with albumin serves as a model of the complex of Gemcitabinel with albumin and allows visualization of the complex spread within the tumor. Methods and Results
Nude (athymic mice) (.about.5 weeks of age) were injected subcutaneously with a cell suspension containing approximately 10.sup.7 cells/0.1 ml of human mammary tumor cell line MCF7. On Day 28 following tumor cell implantation, all tumors were measured as described below, and the measurement recorded for each mouse as the pre-treatment baseline tumor volume. Tumor measurement were performed using calipers, to measure the tumor in two dimensions, at approximately 90.degree.. to each other, at the longest and widest points. The tumor volume was calculated according to the formula, (W.sup.2.times.L)/2, where W is the tumor measurement at the widest point, and L is the tumor dimension at the longest point. Mice with tumor volumes within the range of 5-8 grams were allocated to the study. The mice were injected intratumorally with 1 ml of a solution of Evan's blue albumin in buffered saline using a Sage Instrument Model #355 syringe pump. The albumin dye complex serves as a visual model for the albumin Gemcitabinel complex. The solution was injected into the tumor at various flow rates between 0.0167 ml/min to 0.5 ml/min which corresponded to various back pressures. The faster the flow rate the higher the (not measured) back pressure is presumed to be. The flow rates tested were:
Flowrate „^^__
0.0167 ml/min (t ml/60min)
0.05 ml/min (1 ml/20min)
0.1 ml/min (1 ml/lOmin)
0.2 ml/min (1 ml/5min)
0.5 ml/min (1 ml/2 min)
After the injections the mice were sacrificed, the tumor removed and the extent of the spread of the blue dye in the tumor measured visually.
From the results are given in the following table. One can see that the raising of the pressure results in a more efficient spread of the dye.

Flow rate Percent of tumor volume dyed
0.0167 ml/min (1 ml/60 min) 2-5
0.05 ml/min (1 ml/20 min) 20-40
0.01 ml/min (1 ml/10 min) 40-60
0.2 ml/min (1 ml/5 min) 70-90
0.2 ml/min (1 ml/2 min) >90
Conclusion
The results exemplify that the albumin can be effectively spread within the entire tumor volume when the pressure of the infusion is slightly raised. In our system, a flow rate of 0.2 ml/min suffices to raise the pressure and spread the soluble albumin complex. The efficient spread of the Gemcitabinel albumin complex results in more efficacious treatment of the solid tumors. EXAMPLE 2
In Vivo Evaluation of the Anti-Tumor Effect of Intratumoral Injections of Gemcitabinel/HSA in Human Breast Tumor (Cell line MCF7) Xenografts in Nude Mice, Purpose of Study
The purpose of the study is to assess the anti-tumor effect of intratumoral injections of Gemcitabinel/HSA, a novel proprietary compound of Gemcitabinel complexed with human serum albumin) against a human mammary tumor xenograft (cell line MCF7) in immunodeficient mice. The potential of Gemcitabinel/HSA to reduce tumor size is compared to the standard chemotherapeutic agent, TAXOL.RTM. Methods and Results
There are five study groups containing 6-10 mice per group. The mice are allocated to the 5 groups.
Nude (athymic mice) (.about.5 weeks of age) are injected subcutaneously with a cell suspension containing approximately 10.sup.7 cells/0.1 ml of human mammary tumor cell line MCF7. The mice are examined routinely for the appearance of tumors. On Day 28 following tumor cell implantation, all tumors are measured as described below, and the measurement recorded for each mouse as the pre-treatment baseline tumor volume. Tumor measurement are performed using calipers, to measure the tumor in two dimensions, at approximately 90.degree. to each other, at the longest and widest points. The tumor volume are calculated according to the formula, (W.sup.2.times.L)/2, where W is the tumor measurement at the widest point, and L is the tumor dimension at the longest point. All mice with tumor volumes within the range of 5-8 grams are allocated to study groups. Allocation to treatment groups are carried out based on the volume of the individual tumors,

with each study group receiving an approximately equal representation of all tumor volumes. At study baseline, Day "0" of the Treatment Phase, all mice are receive the first injection according to their study group assignment. Approximately twenty-three hours later, the tumors are measured as described above, and the volumes recorded. Immediately following measurement, within 24 hours of the first injection, the mice receive a second injection according to the study group assignment. Post-treatment tumor volumes are assessed at 48 hours, 7 days, 14 days, and 21 days following the initial injection.

Group
Number Drug Dosage Method of Administration Number of
Injections
(within 24
hours)
I No treatment (control) - - -
11 Saline (control) 0.2 m]/gma Intratumoral 2
111 TAXOL ® 0.2 ml/gma Intratumoral 2
IV Gemcitabinel/HAS 0.2 m!/gma Intratumoral 2
V Gemchabinel/HAS 0.2 mi/gma Intratumoral (via high-pressure
infusion) 2
aper gram tumor weight at 1 mg Gemcitabinel/ml
The mice are sacrificed and the tumors removed and weighed. The final weights for each treatment group are averaged and compared to the final weights obtained for the "no-treatment" group.
For each mouse within a study group, the post-treatment tumor volumes just before the 2.sup.nd injection at 24 hours, and at 48 hours, 7, 14 and 21 days following the initial injection, are measured and recorded. The relative tumor volume (post-treatment tumor volume/pre-treatment baseline tumor volume) are recorded at each time point, and the mean relative tumor volume for each time point, for all mice within a study group, are determined. Additionally, following sacrifice, the final weights for the tumors for each study group are averaged and compared to the final weights observed for the "no-treatment" group.
The expected results of the measurement of relative tumor volume (100.times.post-treatment tumor volume/pre-treatment baseline tumor volume) (expected results) are tabulated in the following table:

Group % tumor
volume at 2
days % tumor
volume at 7
days % tumor volume at 14
days % tumor
volume at 21
days
I 105 125 150 175
II 105 125 150 170
III 50 50 75 85
IV 40 40 60 75
V 40 25 25 40
Conclusion
Intratumoral injections of soluble Gemcitabinel/HSA are an effective method of affording tumor shrinkage. Two intratumoral injections separated by 24 hours are effective in shrinking the tumor to about 40% of its original value. Elevated pressure makes the injections more effective. Further injections, in an improved protocol, could conceivably bring about a full remission in the tumor.

We claim:
1. A process of administering Gemcitabinel, comprising; introducing an intratumoral dose of a Gemcitabinel formulation; and, subsequently providing an initial intravenous infusion of Gemcitabinel about 24 hours to about 7 days after the intratumoral dose.
2. The process as claimed in claim 1, wherein the Gemcitabinel formulation is a mixture of Gemcitabinel and plasma protein in an amount effective to solubilize the Gemcitabinel,
3. A process of administering Gemcitabinel to inhibit uncontrolled growth of cell
comprising: administering at least one intratumoral dose of a first formulation
comprising Gemcitabinel and a plasma protein, thereby inducing apoptosis; and
administering intravenously to a patient at least one dose of a second formulation
comprising Gemcitabinel, the first dose of the second formulation occurring about 1
to about 7 days after the the intratumoral dose, wherein the first and second
formulations are the same or different, and wherein the amount of plasma protein in
the first formulation is an amount effective to solubilize the Gemcitabinel.
4. The process as claimed in claim 3, wherein the plasma protein is selected from the group consisting of human serum albumin and .gamma.-globulin.
5. The process as claimed in claim3, wherein the dose of Gemcitabinel formulation is between about 1 to about 60 mg of Gemcitabinel.
6. The process as claimed in claim 3, wherein the Gemcitabinel formulation is between
about 4 to about 10 mg/ml Gemcitabinel.
7. The process as claimed in claim 3, wherein the intratumoral dose is administered by a plurality of injections of the Gemcitabinel formulation.
8. The process as claimed in claim 3, wherein the intratumoral dose of the Gemcitabinel formulation is administered by syringe pump.
9. The process as claimed in 3, wherein the intravenous infusion of Gemcitabinel comprises administering between about 70 to about 280 mg of Gemcitabinel.
10. The process as claimed in 3, wherein the intravenous infusion of Gemcitabinel comprises administering between about 100 to about- 200 mg/m.sup.2 of Gemcitabinel.
11. The process as claimed in claim 3, wherein the intravenous infusion of Gemcitabinel comprises administering between about 135 to about 175 mg/m.sup.2 of Gemcitabinel.

12. The process as claimed in claim 3, wherein the intravenous infusion of Gemcitabinel
comprises administering a mixture of Gemcitabinel arid plasma protein in an amount
effective to solubilize the Gemcitabinel.
13. The process as claimed in claim 12, wherein the solubilizing plasma protein is selected from the group consisting of human serum albumin and .gamma.-globulin.
14. The process as claimed in 1, wherein the intravenous infusion of Gemcitabinel comprises administering Gemcitabinel and polyoxyethylated castor oil.
15. The process as claimed in any preceding claim, wherein the additional intravenous dose is administered about 4 to about 21 days subsequent to the intravenous infusion.