WO2010008561A2

WO2010008561A2 - Methods and compositions for protecting against cytotoxic therapy

Info

Publication number: WO2010008561A2
Application number: PCT/US2009/004110
Authority: WO
Inventors: Scott Chappel
Original assignee: Tokai Pharmaceuticals, Inc.
Priority date: 2008-07-16
Filing date: 2009-07-16
Publication date: 2010-01-21
Also published as: WO2010008561A3

Abstract

The present invention is directed to methods and compositions of therapy. More particularly, the methods and compositions disclosed herein form an adjunct to chemotherapy, radiation therapy and other therapies that cause myelosuppression. The methods of the invention use PTHrP or analogs thereof to prevent, reduce, abrogate or otherwise reverse radiation-induced or drug-induced or antibody-induced cytotoxicity, and are especially applicable to protection of hematopoietic cells against radiation and/or chemotherapy-induced cytotoxicity.

Description

METHODS AND COMPOSITIONS FOR PROTECTING AGAINST CYTOTOXIC THERAPY

BACKGROUND

Field of the Invention The present invention is directed to methods and compositions for improved therapeutic methods that use cytotoxic agents. More specifically, the invention is directed to the use of PTHrP or analogs thereof to prevent, reduce, abrogate or otherwise reverse radiation-induced or drug-induced or antibody-induced cytotoxicity, especially to hematopoietic cells. Background of the Related Art

Many different tumors are treated with combination chemotherapy or radiation therapy. However, the efficacy of these therapies is greatly hindered by the fact that these therapies have a cytotoxic effect on the bone marrow and the hematopoietic cells of the body. In almost all instances, there is a dramatic reduction in circulating red and white cells and platelets. These reductions restrict the dose and limit the potential benefit of these therapeutic regimens. Further, the risk of infection is directly related to the severity of myelosuppression induced by the cytoxic treatments. Despite the use of broad spectrum antibiotics, sepsis is not infrequent and the morbidity associated with nonfatal infection is substantial. Reductions in circulating levels of red blood cells also results in dramatic fatigue and anorexia which can further complicate recovery. The severity of myelosuppression also limits the dose and duration of exposure to chemotherapy or radiation therapy which may compromise the effectiveness of the therapy's tumor killing capability.

Following completion of a chemotherapy or radiation therapy regimen, changes in the patients blood cell populations are routinely monitored to begin treatment with stimulators of red and white blood cell proliferation. During treatment and follow-up, monitoring the patient's blood cell count is important (e.g., prior to each treatment cycle and 10-14 days after each treatment cycle).

During the examination of a patient following chemo- or radiation therapy, a complete blood cell count and other blood chemistries are tests that are routinely performed. Patients that exhibit symptomatic anemia may be immediately transfused with packed red blood cells and platelets. Cytopenias (anemia, neutropenia and thrombocytopenia) are clearly of great concern post-therapy. Hematopoeitic growth factors such as G-CSF, GM-CSF and erythropoietin are given immediately following chemotherapy in an effort to protect the patients from opportunistic infections, sepsis and associated morbidity and mortality. Growth factor support is provided as needed.

In order to effect hematopoietic reconstitution early-term and long-term, it is important to administer G-CSF, GM-CSF, or PEGylated G-CSF as soon as possible post- radiation or chemotherapy exposure. Nevertheless, the effect of supportive care and CSF therapy on long-term immune reconstitution is unknown in the context of severely irradiated animals. There is, however, a substantial clinical database showing that the multicycle, myelosuppressive chemotherapy or myeloablative conditioning prior to stem cell transplant may have deleterious effects on immune recovery. Neutropenia is common in patients with cancer who receive myelosuppressive chemotherapy and contributes to morbidity associated with cancer. Neutropenia is associated with increased risk of infections which can be life threatening and require aggressive treatment with intravenously administered antibiotics. Such antibiotic therapy necessitates hospitalization and may be associated with other complications. Active management of neutropenia is thus of substantial importance in patients undergoing cytotoxic chemotherapy for the treatment of neoplastic disease.

Many studies have shown that recombinant G-CSF increases WBC counts and decreases the duration of neutropenia, days of hospitalization and number of infections. This therapeutic intervention also results in a decrease in chemotherapy delays, dose reductions and more patients receiving the full regimen of treatment. However, treatment of patients with filigrastim prior to chemotherapy resulted in a dramatic reduction in the marrow repopulation capacity due to the large number of mitotically activate cells within the marrow that are sensitive to chemotherapy. This resulted in a dramatically reduced overall regenerative capacity of the marrow post chemotherapy. In addition, it has been previously reported that repeated cycles of therapy with G-CSF following chemotherapy as a restorative regimen after cyclic chemotherapy may seriously reduce marrow repopulating ability or stem cell capacity of the patient. Excessive stimulation of primitive stem cell proliferation by cytokines may lead to a loss of primate stem cells and premature bone marrow failure. Studies in mice have shown that administration of G-CSF to speed recovery from repeated doses of cyclophosphamide damages primitive stem cells. It is assumed that G-CSF induced damage of primitive stem cells after multiple doses of cytotoxic agents is the increased proliferation of stem cells in response to G-CSF at the expense of self-renewal. Marrow that is already compromised appears to be most damaged by multiple exposures to G-CSF. To date, there is no effective treatment for the prolonged T cell deficiencies associated with cytotoxic therapy. There is a significant delay in regeneration of CD4+ T cells which leads to an imbalance in the ratio of CD4/CD8 cell, this, along with a limited T-cell receptor repertoire leave the patient at risk for opportunistic infections. Long term immune reconstitution requires regeneration of naϊve, thymic-dependent T-cell. Several cytokines including IL-2, IL-4, IL-7, IL- 17, c-kit ligand, flt-3, thymic stromal lymphopoietin, and keratinocyte growth factor are associated with T-cell differentiation, proliferation, and enhanced thymopoiesis and functional recovery of peripheral T cells. Exemplary hematopoietic factors that could be employed for hematopoietic reconstitution and for enhancing recovery of thymopoiesis and immune reconstitution include but are not limited to Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-10, IL-1 1, IL- 12, IL-13; IL-15, IL- 16, IL- 17, stem cell factor (also known as c-kit ligand), and M-CSF.

Despite the use of cytokines as a post-irradiation or post-chemotherapy reconstitution of immune cells, however, there is a continued need for further methods of preventing, mitigating or reversing toxicity to myeloid and hematopoietic cells, which is a limiting side effect of treatment of various diseases in subjects with cytotoxic agents. SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions of therapy that can be used as an adjunct to chemotherapy, radiation therapy and other therapies that cause myelosuppression. The methods of the invention use PTHrP or analogues thereof to prevent, reduce, abrogate or otherwise reverse radiation-induced or drug-induced or antibody-induced cytotoxicity, and are especially applicable to protection of hematopoietic cells against radiation and/or chemotherapy-induced cytotoxicity.

In certain preferred aspects of the invention there are described methods of decreasing chemotherapy and/or radiation therapy-induced cytopenia in a subject comprising administering to the subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof, wherein the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in an amount effective to decrease cytopenia (including but not limited to anemia, neutropenia and thrombocytopenia) in the subject as compared to the cytopenia induced in the absence of the administration.

The administration of the composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof may be carried out prior to, after or subsequent to the chemotherapy and/or radiation therapy. In particular embodiments, it is contemplated that the administration of the composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is carried out prior to the chemotherapy and/or radiation therapy. For example, the composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered to the subject between 4-7 days prior to initiation of cytotoxic chemotherapy and/or radiation therapy. Thus, the PTHrP, a PTHrP fragment, a PTHrP analogue, or a derivative thereof is advantageously employed as a prophylactic composition.

The methods of the invention also may be used to decrease myelosuppression in a subject comprising administering to the subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof. In specific embodiments, the myelosuppression is produced by chemotherapy or radiation therapy or immunosuppressive therapy received during organ transplantation. In such methods, the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is preferably administered prior to chemotherapy or the radiation therapy.

Also contemplated herein are methods of decreasing the dose or duration of chemotherapy or radiation therapy to be administered to a subject in need thereof comprising administering to the subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof.

Another embodiment of the invention contemplates a method of preventing or decreasing the level of reduction in circulating blood cells in a subject receiving radiation therapy and/or chemotherapy, comprising administering to the subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof prior to administration of the radiation therapy and/or chemotherapy, wherein administration of the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof prevents or produces a lower level of reduction of circulating blood cells in the subject as compared to a subject that does not receive the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof.

The methods of the invention also may be used to treat a subject that is at risk of having a decrease in the number of cells of the hematopoietic system, comprising the step of administration to the subject an effective amount of PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof, wherein the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered before, after, or during the chemotherapy and/or radiation therapy. For example, the cells the hematopoietic system may be selected from the group consisting of, including but not limited to T cells, B cells, granulocytes, macrophages, mesenchymal cells, osteoblasts, osteoclasts and multipotent adult progenitor cells, or combinations thereof. In specific embodiments, the decrease in the number of cells of the hematopoietic system is associated with chemo- and/or radiotherapy and/or removal of blood progenitor cells. For example, this may occur as a result of the chemotherapy and/or radiotherapy and/or removal of blood progenitor cells being administered to treat cancer.

Also contemplated is a method for protecting hematopoeitic cells from cell death in response to a chemotherapeutic and/or radiation comprising contacting the cells with a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof prior to contacting the cells with the chemotherapeutic agent and/or radiation.

The invention further is directed to a method for treating a cytotoxic agent-induced hematopoietic or myeloid toxicity in a human patient which comprises administering prior to, simultaneous with or subsequent to the administration of the cytotoxic agent, a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to prevent, mitigate or reverse such hematopoietic or myeloid toxicity. In such methods, the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof may, but need not necessarily be administered in combination with one or more hematopoietic factors selected from the group consisting of Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-12, IL-13; IL-15, IL-16, IL-17, stem cell factor (c-kit ligand), and M-CSF (including PEGylated derivatives and biologically fragments thereof) is administered in an amount effective to prevent, mitigate or reverse hematopoietic or myeloid toxicity.

It should be noted that the one or more hematopoietic factors administered in the combination adjunct therapy immediately above may be administered prior to, concurrently with or subsequent to the administration of PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof. Also provided is a method of treating cancer therapy in a mammalian patient which comprises administering to the patient a combination of a cytotoxic agent in an amount effective to treat the cancer and PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to prevent, mitigate or reverse hematopoietic or myeloid toxicity, wherein the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered prior to, simultaneously with or subsequent to the administration of the cytotoxic agent, wherein administration of the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof reduces the dose of the cytotoxic agent needed to produce a therapeutic effect in the patient. In some embodiments, the cytotoxic agent is a therapeutic agent, including but not limited to radiation therapy or chemotherapy.

Also taught herein is a method treating cancer therapy in a mammalian patient which comprises administering to the patient a combination of a cytotoxic agent in an amount effective to treat the cancer and a combination of PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof and at least one hematopoietic growth factor in an amount effective to prevent, mitigate or reverse hematopoietic or myeloid toxicity, wherein the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered prior to the administration of the cytotoxic agent.

The methods of treating cancer may be used in a mammalian patient is suffering from a cancer selected from the group consisting of Hodgkins lymphoma, NonHodgkins lymphoma, pancreatic cancer, melanoma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, lung cancer, Kaposi's sarcoma, leukemia, lymphoma, gastric cancer, colon cancer, colorectal cancer, esophageal cancer, renal cancer, and head and neck cancer. In the methods of treating cancer, the hematopoietic growth factor may be selected from the group consisting of Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-13; IL- 15, IL- 16, IL- 17, stem cell factor (c-kit ligand), and M-CSF (including PEGylated derivatives and biologically active fragments thereof).

Also taught herein is a method for preventing or mitigating myelosuppression in a human patient undergoing therapy for cancer or tissue or organ transplantation, which comprises administering to the patient a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to treat myeloid toxicity, wherein the cytokine is administered prior to, simultaneously with or subsequent to the therapy.

The invention may be used as a method for preventing or mitigating hematopoietic cell depression in a human patient undergoing therapy for cancer or tissue or organ transplantation which comprises administering to the patient a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to treat myeloid toxicity, wherein the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered prior to, simultaneously with or subsequent to the administration of the therapy.

Further aspects of the invention contemplate a method for treating a cytotoxic agent-induced hematopoietic or myeloid toxicity in a human patient which comprises administering prior to, simultaneous with or subsequent to the administration of the therapeutic agent, PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof and a hematopoietic factor in an amount effective to prevent, mitigate or reverse such hematopoietic ar myeloid toxicity. For example, such a cytotoxic agent may include a radionuclide, including but not limited to Iodine- 131, Strontiun-89, Yttrium-90, Rhenium 186, Rheniuml88, Cobalt 60; Cesium 137; Iridium 192, and Radium 226. In some embodiments, the cytotoxic agent is conjugated to an antibody or a bone-seeking chemical. Exemplary bone-seeking chemicals include orthophosphate or diphosphonate.

Also provided herein is a method of alleviating a physical symptom caused by hematopoietic or myeloid toxicity in a human patient undergoing therapy with a cytotoxic agent, wherein the patient is administered prior to, simultaneous with or subsequent to the administration of the cytotoxic agent, a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to prevent, mitigate or reverse such hematopoietic or myeloid toxicity. For example, the physical symptom may be bone pain associated with bone cancer or bone metastasis.

In the methods of the present invention, an exemplary patient is a cancer patient. For example, the subject has a cancer selected from the group consisting of Hodgkins lymphoma, Non-Hodgkins lymphoma, pancreatic cancer, melanoma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, lung cancer, Kaposi's sarcoma, leukemia, lymphoma, gastric cancer, colon cancer, colorectal cancer, esophageal cancer, renal cancer, and head and neck cancer. In some embodiments, the patient is a patient receiving an organ transplant and receiving immunosuppressive therapy to kill T-cells. The PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof may be administered through any typical route of administration including, but not limited to administration via subcutaneous, intravenous, intradermal, intraarterial, intramusclar, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term release), aerosol, sublingual, nasal, anal, vaginal, oral, or transdermal delivery, or by surgical implantation, or a combination thereof.

In some embodiments, the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in a single dose. In other embodiments, the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in multiple doses or as a continuous infusion during therapy. In specific aspects of the invention, the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in a dose of about 0.5 μg/kg body weight to about lOμg /kg body weight of subject/day.

The PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof may be administered on each of 2, 3, 4, 5, 6 or 7 days prior to the administration of chemotherapy or radiation therapy. As noted herein throughout, the methods contemplate that the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof may be administered in combination with one or more hematopoietic factors. The hematopoietic factor may be selected from the group consisting of Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, 1L-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-I2, IL-13; IL-15, IL-16, IL-17, stem cell factor (c-kit ligand), and M-CSF or combinations thereof. In addition the hematopoietic factor may be a biologically active fragment of such a factor. The hematopoietic factor or fragment thereof also may be PEGylated.

In certain specific embodiments, the composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof comprises PTHrP 1-34, or an analogue thereof. An exemplary PTHrP 1-34 analogue is semparatide.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF DRAWINGS

Figures 1, 2 and 3 depict the hematoprotective effect of Semparatide administered to mice for 14 days prior to administration of a high dose of 5-fluorouricil, showing higher levels and faster recovery post chemotherapy of circulating WBCs, lymphocytes and neutrophils respectively.

DETAILED DESCRIPTION OF INVENTION

There is a need to establish methods and compositions for use in subjects undergoing chemotherapy and/or radiation therapy in order to protect, prevent, mitigate, reduce, abrogate, or otherwise reverse cytopenia (e.g., anemia, neutropenia and thrombocytopenia) produced by the chemotherapy and/or radiation treatment, particularly a need for strategies to increase stem cell populations prior to chemotherapy and/or radiation therapy that do not result in a chemotherapy-induced and/or radiation therapy-induced reduction in overall stem cell and WBC regenerative capacity.

Administration of PTHrP, prior to or immediately after a chemotherapeutic insult will decrease the reduction in the population of hematopoietic cells and thus decrease the cytoreduction that is normally observed after such ablative therapies. By decreasing the period of vulnerability to opportunistic infections post therapy, patients can return to normal life sooner, spending less time in an intensive care unit waiting for more conventional therapies to increase the population of white and red cells and platelets. The hematopoietic stem cell niche is intertwined with osteoblasts and bone stroma.

Bone marrow stroma and local osteoblasts permit hematopoiesis by providing environmental cues, physical connections and the appropriate growth factor support. Osteoblasts and stromal cells are able to support hematopoietic progenitors and allow them to expand. The interaction between these cells has been called a niche. Without intending to be bound by theory, it is believed that stimuli that increase the number of osteoblasts, increase the availability of hematopoietic stem cell niches, which would increase the number of hematopoietic stem cells available for release into the systemic circulation following stimulation (injection of G-CSF or administration of chemotherapy). More recently, it has been shown that osteoblasts secrete cytokines such as G-CSF, GM-CSF, ILl, IL6 and LIF all of which have been shown to modulate hematopoiesis. Indeed, the intersection of a number of recent observations suggests that a physical "harbor" is created by bone marrow stroma and osteoblasts which provides a niche for hematopoietic stem cells to attach and proliferate. These cells are held in place by cell to cell adhesion. When activated by G-CSF or other stimuli such as chemotherapy, proteases (specifically MMP-I) are activated and free the blood cell so that it may migrate to the vascular network within the marrow and move to the peripheral vascular system.

Activation of osteoblasts within the stem cell niche of bone marrow allows for the proliferation and migration of stem cells to that niche. Upon entry into the niche, these cells are tethered and following this physical attachment and through the activation of membrane protein ligand and receptor, Notch and Jagged, remain in the G₀ resting state and are thus not sensitive to the cytotoxic effects of chemotherapy. Upon activation of the stem cell niche, either by the injection of G-CSF or administration of chemotherapy, many more cells are available to enter into the differentiation pathway. Thus, greater numbers of mature WBCs and neutrophils are released into the peripheral circulation, thus decreasing the duration and extent of neutropenia. In addition, due to the more physiological method of leukocytosis, no depletion of marrow regenerative capacity is experienced. PTHrP, e.g. Semparatide, is believed to increase the hematopoietic pool size by a number of mechanisms:

• strong stimulation of the growth of trabecular bone which enlarges the hematopoietic stem cell niche

• promote the attachment of stem cells by elaborating growth factors and "mooring lines" for cellular attachment • directly stimulate stem cells to differentiate by the elaboration of growth factors

• induce the expression of ligands and receptors (such as Jagged and Notch) which allow for intercellular communication and survival of hematopoietic stem cell differentiation.

The therapeutic methods of the invention are useful for reconstitution of levels of all blood cell types after such cytotoxic therapy. In the present invention, it is shown that PTHrP, and fragments, and analogs thereof, may be used to avoid the cytopenia observed in patients following chemo- or radiation therapy. Due to the unique property of this peptide to stimulate an intracellular process in hematopoietic progenitor cells that provides them with the capacity to avoid programmed cell death (apoptosis), in certain specific embodiments, it is proposed that the subject may be pretreated with the PTHrP or analog thereof prior to the initiation of the cytotoxic chemotherapy or radiation therapy in order to avoid the myelosuppression and associated cytopenias that limit the dose and duration of chemotherapy treatment.

Prophylactic treatment with PTHrP will also be useful in that the period of time during which the patient is at risk for developing opportunistic infections and sepsis can be greatly reduced. The use of PTHrP or analogs thereof as described herein will also advantageously avoid the need for transfusions of RBC and platelets. It is envisioned that this prophylactic therapy would also avoid the need for expensive treatments with blood cell growth factors such as erythropoietin and G-CSF. However, it is contemplated that in some circumstances it may be desirable to combine the treatment with PTHrP or analog thereof, with treatments that use blood cell growth factors in order to achieve a more effective recovery than that achieved in the absence of application of the PTHrP.

Thus, in preferred aspects of the invention, the use of PTHrP, fragments or analogs thereof is used to improve the therapeutic efficacy of anticancer, antimicrobial and autoimmune disease, and anti-organ rejection therapy in that the PTHrP, fragments, or analog thereof is used to prevent, mitigate or reverse adverse radiation-induced or drug-induced toxicity, especially to hematopoietic cells. Use of the PTHrP, fragment, or analog thereof will allow the subject to tolerate higher doses of cytotoxic agents that are administered to the subject for therapeutic purposes.

The PTHrP, fragment, or analog thereof may also increase the duration of time during which the cytotoxic agents can be administered and the dose-limiting hematopoietic cell toxicity that is characteristic of such cytotoxic agents can be prevented, palliated or reversed using adjunct therapy with PTHrP. The present invention is directed to methods of treatment in which PTHrP is administered to a subject that has or is at risk of developing cytopenia. Typically, the subject is one in which the cytopenia will be induced as a result of cytotoxic agents such as chemotherapy, radiotherapy, antimicrobial agents and the like. The invention contemplates methods of treating such subjects in which PTHrP or an analog thereof or a combination of PTHrP and an analog thereof is administered to prevent, mitigate, reduce, abrogate, reduce or otherwise reverse radiation-induced or drug-induced toxicity of normal cells, and more particularly, hematopoietic cells.

Throughout the specification and the claims herein the term "PTHrP" is used. It should be understood that these methods may use natural wild-type PTHrP, a natural variant of PTHrP, an analog of PTHrP or a derivative of PTHrP that has a structure based on PTHrP but has been modified to contain a moiety that increases the uptake or distribution characteristics of the PTHrP or otherwise improve the therapeutic properties of the peptide. PTHrP and its various permutations that may be used in the present invention are described in further detail below, however, it should be understood that any of the various forms of PTHrP, its analogs or its derivatives may be used in the present methods.

In essence, certain embodiments of the invention are directed to methods that provide an adjunct therapy in which PTHrP (including its analogs and derivatives) is used as the adjunct that allows the administration of higher doses of cytotoxic agents due to increased tolerance of the recipient mammal. Moreover, adjunct PTHrP therapy can prevent, palliate, or reverse dose-limiting cytotoxic effects of the cytotoxic agents on the hematopoeitic cells.

The PTHrP also has a radioprotective effect on hematopoietic cells. A great deal of therapeutic administration of radioisotopes involves administration of beta emitters, alpha emitters and/or generation of radioisotope in situ by neutron activation of Boron-10 atoms (resulting in alpha emission from the unstable nuclide produced by neutron absorption.) The present invention contemplates administration of PTHrP (or analog or derivative thereof) to the subject either prior to, during or after application of the radiotherapy.

Prior to the present invention, it could not be predicted with accuracy that drug- or toxin-induced hematopoietic or myeloid toxicity would respond to PTHrP treatment in humans. However, given the teachings of the present invention, it is now possible to administer not only PTHrP, but also related molecules, such as PTHrP analogs or derivatives in order to achieve a radioprotective or chemoprotective effect on hematopoietic cells. The administration of the PTHrP or related molecule may be prior to the administration to the chemotherapy, radiotherapy or other drug or toxin-based therapy, and as such, can be a prophylactic administration of the agent. Alternatively, the PTHrP or related molecule may be administered simultaneously or subsequent to the chemotherapy, radiotherapy or other drug or toxin-based therapy to mitigate or reverse myeloid or hematopoietic toxicity.

It should be understood that the methods of the invention may be particularly useful as adjuncts to conventional anti-cancer therapies in which the subject is administered one or more tumoricidal agent, e.g., a drug and a radioisotope, or a radioisotope and a Boron- 10 agent for neutron-activated therapy, or a drug and a biological response modifier, or an antibody conjugate and a biological response modifier. The PTHrP can be integrated into such anti-cancer therapeutic regimens to maximize the efficacy of each component thereof.

In some examples, it has been shown that certain anti-leukemic and anti-lymphoma antibodies conjugated with beta or alpha emitting radioisotopes can induce myeloid and other hematopoietic side effects when these agents are not solely directed to the tumor cells, particularly when the tumor cells are in the circulation and in the blood-forming organs. The methods of the invention contemplate administration of the PTHrP or related molecule prior to, and/or concomitantly with and/or subsequent to the administration of such antibody-based therapies to reduce or ameliorate the hematopoietic side effects of the anticancer agent, while allowing the beneficial anticancer effect of the agent. Thus, depending on the specific condition being treated, an appropriate dose of the

PTHrP or analog or derivative can be administered prior to, simultaneously with or subsequent to the administration of the therapeutic agent. It is desirable to maximize the cytotoxic activity of the therapeutic agent on the pathological lesion, such as cancer cells or infectious organisms, while minimizing toxicity of that agent to the myeloid and other hematopoietic cells. In some circumstances, the PTHrP adjunct therapy may be administered continuously with the cytotoxic therapeutic agent in order to achieve the most beneficial hematoprotective effects of the PTHrP.

As noted above, numerous anticancer therapies produce a deleterious myelosuppressive effect. Typical anti-cancer agents include biological agents (biotherapy), chemotherapy agents, and radiotherapy agents. Generally, these compositions are provided in a combined amount effective to kill or inhibit proliferation of the cancer cell, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer. In doing so, these therapies typically also have an effect on normal, non cancer cells. The present invention is particularly concerned with the deleterious effects of such therapies on normal hematopoeitic cells. The cancer cells are generally contacted with the anticancer agent and at the same time, the hematopoietic cells also come into contact with such agents.

Myeloid cell death in response to chemotherapy and radiotherapy agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy by combining it with agents that are myeloprotective or are able to reconsistitute myeloid cells. For example, cytokines are typically administered after the chemotherapy and or radiotherapy to facilitate myeloid reconstitution. In the context of the present invention, it is contemplated that PTHrP and related compositions could be used in conjunction with chemotherapeutic, radiotherapeutic, immunotherapeutic or other biological intervention, in order to act as myeloprotectant and to enhance the growth of hematopoeitic cells.

PTHrP may be administered prior to, after or during the administration of the other therapeutic agent intervals ranging from minutes to weeks. In embodiments where the other agent and the PTHrP are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

The hematoprotection afforded by the PTHrP is protection against any conventional chemotherapy, including use of for example, alkylating agents (cyclophosphamide), anti metabolites (azothioprine), plant alkaloids and terpinoids (vinblastine, etoposide, and paclitaxel), topoisomerase inhibitors (topotecan) as well as hormone therapy (antiestrogen: tamoxifen; antiandrogens: bicalutamide). Thus the PTHrP compositions may be used in order to protect the subject from the myelosuppressive effects of exemplary agents that include but are not limited to cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, Temazolomide (an aqueous form of DTIC), or any analog or derivative variant of the foregoing. Those skilled in the art will understand that these are merely exemplary chemotherapeutic agents and that the PTHrP-based methods of the invention can be used to protect against the myelosuppressive effects of other such chemotherapy agents. The hematoprotection derived from PTHrP administration can also be used to protect hematopoietic cells from other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Such radioisotopes include, but are not limited to Iodine¹³¹, Strontium⁸⁹, Yttrium⁹⁰, Rhenium¹⁸⁶, Rhenium¹⁸⁸, Cobalt⁶⁰; Cesium¹³⁷; Iridium¹⁹², and Radium²²⁶. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic agent (e.g., the PTHrP or the cytokine or an expression construct that encodes such an agent) and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve effective therapy, the PTHrP and other hematoprotectant is delivered to a cell in a amount effective to protect the cell from cell death in response to chemo- or radiotherapy.

Throughout the treatment protocols, it may be useful to monitor the white blood cell (WBC) count, Red blood cell (RBC) count, platelets and other blood elements in the subject. Thus, it is contemplated that as part of the treatment regimen the subject's blood sample will be monitored for components, including but not limited to erythrocyte (red blood cell/RBC) count, thrombocyte (platelet) count, and including a differential WBC analysis to monitor the myloid/lymphoid series, as well as the bone marrow hematological picture during the course of therapy. It will be particularly desirable to assess the subject for possible depletion of myeloid lymphoid forms, but also the status of immature erythrocytes, myelocytes, lymphocytes and thrombocytes. Monitoring these parameters will permit optimization of the PTHrP adjunct treatment with the cytotoxic agent therapy. Depending upon which hematologic element is adversely affected, the amount and duration of the PTHrP and cytotoxic agent can be tailored to each circumstance.

Correlation of the choice of the amount of PTHrP and its combination with another hematoprotectant (such as a cytokine as discussed below), and doses thereof, to hematotoxicity is important, since each therapeutic regimen involving a cytokine generally will have its effect mostly on particular hematopoietic elements. For example, if a cytotoxic agent has both severe myeloid and thrombocytic toxicity, the combination of the PTHrP with IL-I and IL-3 in a composition in which the IL-I and IL-3 are present at a 1:1 or 2:1 (or higher) ratio will be advantageous. Thus, reduction in the WBC count to a level below about 2,000 and platelets to a level below about 20,000 can be reversed by administration of

PTHrP. The administration of the PTHrP can be repeated, with the reversal of the myeloid and platelet depressions occurring within about 5-20 days after the administration of the PTHrP, usually within about 7 days. The ordinary skilled clinician will appreciate that variations in the timing and dosage of PTHrP administration and combinations of the PTHrP with for example cytokines and dosages of these agents are a function of the agent used, the nature of the bone marrow and/or other hematopoietic element depressed, and the nature of the patient (e.g., prior toxicity affecting bone marrow status) and the cytotoxic agent and protocol.

In other embodiments of the invention PTHrP compositions can be administered in order to treat bone pain in patients with bone metastases and primary bone cancers. In such cases, radionuclide therapy has been found to be effective and safe, particularly with the introduction of Sr-89, Y-90 and Re-186 or Re-188, either alone or conjugated to an antibody or a bone-seeking chemical such as orthophosphate or diphosphonate. Chemotherapeutic agents, e.g., 5-fluorouracil (5-FU), have also been known to control bone pain in patients with metastatic carcinoma. P-32-ortho-phosphate can be administered in several ways, including single doses of about 3 to 10 mCi, multiple consecutive doses of about 1.5 mCi, or multiple intermittent doses of 7 to 10 mCi as clinically required. In multiple and intermittent dose schedules, total doses can range from 5 to 20 mCi, depending on patient response and side effects. In order to reduce the myelosuppression of this treatment and increase the effects against bone pain and possibly also inhibit tumor growth, these doses can be increased by from about 10% to about 35%, preferably 15 to 25%, by simultaneous administration of continuous or intermittent doses of about 5 to 20 μg of IL-I, more preferably single repeated dose of about 0.5 μg PTHrP/kg body weight to about lOμg PTHrP/kg body weight of subject/day PTHrP (including full-length PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof) either alone or in combination with a cytokine, thereby extending the time over which radionuclide therapy can be tolerated by the patient. Similarly, Re-186- diphosphonates can be used for bone pain palliation in single doses of about 5 to 10 mCi, repeated up to three times, in combination with administration of the PTHrP either prior to and/or, simultaneously and/or post-therapy with Re-186-diphosphonates. Again such administration of the PTHrP may be in combination with a cytokine which can be administered prior to and/or simultaneously and/or post-administration of the diphosphonates and/or the PTHrP. The therapy with the composition comprising the PTHrP and/or the composition comprising the cytokine can be repeated several times during a 1-2 week therapy regimen.

1-131 is another effective radioisotope that has been used for the treatment of cancer. It has been shown to be especially useful for treating primary and metastatic, well- differentiated thyroid carcinomas. A dose of 150 mCi 1-131 has been successful, with most clinicians administering a dose between 100 and 200 mCi. Again however, bone marrow depression is a major complication of this therapy and severely limits the dosages of the I- 131 that can be tolerated by the subject. The present invention contemplates combining 1-131 therapy, using a dose of 150-250 mCi, with PTHrP. Again the PTHrP may be administered alone or in combination with a cytokine. In exemplary embodiments, PTHrP is administered 4-7 days prior to administration of the 1-131 and produces a marked decrease in the myelosuppression seen in the cancer patient in response to the 1-131 compositions. In other embodiments, the PTHrP is administered during or after the 1-131 either alone or in combination with a cytokine and again produces a beneficial decrease in myelosuppression. In some embodiments, the PTHrP-based therapy is continued to one, two, three or more weeks post-radioisotope therapy. Use of the PTHrP leads to the subject having a marked decrease in myelosuppression and also a marked increase in the tolerance of higher doeses of 1-131 doses. If PTHrP therapy is initiated 4-7 days prior to radioisotope administration, and continued twice weekly for 2-3 weeks, doses of 1-131 between 200 and 300 mCi, preferably 200-250 mCi, could be well tolerated. This will therefore lead to an increase in the therapeutic anti-cancer dose of the 1-131 that can be administered. The PTHrP-based therapeutic methods of the invention can be combined with various methods of radionuclide therapy in order to obtain an effective treatment of cancer and other pathological conditions, as described, e.g., in Harbert, "Nuclear Medicine Therapy", New York, Thieme Medical Publishers, 1987, pp. 1-340. A physician experienced in these procedures will readily be able to adapt the PTHrP adjuvant therapy described herein to such procedures to mitigate the hematopoietic side effects of the radionuclides. Similarly, therapy with cytotoxic drugs, either administered alone or as antibody conjugates for more precisely targeted therapy, e.g., for treatment of cancer, infectious or autoimmune diseases, and for organ rejection therapy, is governed by analogous principles to radioisotope therapy with isotopes or radiolabeled antibodies. Thus, the ordinary skilled clinician will be able to adapt the description of PTHrP use to mitigate marrow suppression and other such hematopoietic side effects by administration of the PTHrP before, during and/or after drug therapy.

In addition to being used as an adjunct therapy to chemotherapy and radiation therapy, the PTHrP-based hematoprotection also can be given to subjects being treated with anticancer antibodies. Invading microorganisms and proliferating cancer cells can be targeted with antibodies that bind specifically to antigens produced by or associated therewith. Such antibodies can directly induce a cytotoxic immune response, e.g., mediated by complement, or an indirect cytotoxic immune response, e.g., through stimulation and mobilization of T- cells, e.g., killer cells (ADCC). Certain of such antibodies also produce side effects which include compromise of elements of the hematopoietic system, and such side effects can be prevented, mitigated and/or reversed by administration of PTHrP as described herein. The dose and time period of administration of the PTHrP and/or other hematoprotectant agents (e.g., cytokines as described herein below) will again be correlated to WBC, erythrocyte and platelet counts and other aspects of the status of the hematopoietic system.

The route of administration of the therapeutic agent as well as of the PTHrP should be coordinated and optimized. For example, intracavitary, e.g., intraperitoneal, administration of a radioisotope- antibody conjugate will eventually result in lowering of the blood cell count in a patient given a high dose of the cytotoxic immunoconjugate, due to eventual diffusion of the conjugate into the bloodstream and circulation through the bone marrow. Administration of the PTHrP can be advantageously effected through any typical route of administration that will allow access to the region of greatest hematopoietic cell compromise. Such routes of administration include, subcutaneous, intravenous, intradermal, intraarterial, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term release), aerosol, sublingual, nasal, anal, vaginal, oral, or transdermal delivery, or by surgical implantation at a particular site to have its maximum effect on the circulating peripheral blood cell counts. Intranasal forms of administration of the PTHrP are of particular interest. Cytokines and other hematoprotectants can be administered through the same routes as the PTHrP. The clinician also may determine whether the PTHrP should be administered as a single dose, in multiple doses or as a continuous infusion during the course of therapy. If the PTHrP is administered concomitantly with the cytotoxic agent, it may be given by continuous intravenous infusion over several hours and optionally repeated on one or more days during and after completion of the cytotoxic therapy. Continuous administration of the PTHrP can be effected by any of the transdermal modes of drug administration known to the art or yet to be developed. Similar considerations and routes of administration also should be taken into account for administration of cytokines.

Radioisotopes typically are administered by a variety of routes for cancer therapy. These include, e.g., intravenous, intraarterial, intracavitary (including intraperitoneal), intrathecal and subcutaneous injection, as well as by implantation of seeds of radioactive material at selected sites in the patient's body. The type, extent and time frame for myeloid and other hematopoietic cell toxicity will vary for each mode of administration of the cytotoxic agent and with the type of agent itself. In general, bone marrow toxicity will be the most serious side effect of such therapeutic regimens and intravenous or intraarterial administration of the PTHrP will be the most effective preventive or palliative measure.

Anticancer and antimicrobial, e.g., antiviral, drugs, whose major side effects are hematologic toxicity also are typically administered through any of a variety of routes, including e.g., intravenous, intraarterial, intracavitary (including intraperitoneal), intrathecal and subcutaneous injection. These drugs, including toxins, can be administered systemically without conjugation to a tumor-targeting or infectious lesion-targeting antibody. Marrow toxicity is a common limiting factor in the dosage which can be administered, and the method of the invention is effective in significantly extending the dosage range and duration of therapeutic administration of such agents. The drugs or toxins may be conjugated with antibodies to achieve better targeting of the drug to the pathological lesion in order to improve the therapeutic efficacy of the drugs or toxins. Still further improvement in the therapeutic efficacy of these agents can be achieved by administering these immunoconjugates with PTHrP as contemplated herein. By employing the PTHrP adjunct therapy, it is possible to increase the dosage of the immunoconjugates that can be administered to the subject because the PTHrP affords an increased tolerance for the higher levels of drug, toxin, or other cytotoxic agent necessary for maximal therapeutic activity.

Radioisotope antibody conjugates can be administered by, e.g., intravenous, intraarterial, intracavitary, intrathecal, intramuscular, or subcutaneous routes. Again, intravenous or intraarterial administrations of PTHrP, its derivative or analogs will normally minimize bone marrow toxicity.

In the therapeutic methods, the beta and alpha emitters are preferred for radioimmunotherapy, since the patient can be treated in multiple doses on an outpatient basis. To the extent that the treatment results in bone marrow toxicity, administration of PTHrP either alone or in combination with cytokines can be effected at convenient times by injection or even by transdermal administration of an appropriate level of PTHrP and cytokine.

Boron- 10 compounds has been contemplated as a useful anticancer treatment (e.g., systemic administration of Boron- 10-containing compounds, e.g., borates, carborane compounds and the like, followed by neutron irradiation) however, the acceptance of this therapy has been hampered by the fact that it produces excessive toxicity to normal organs. Attempts to mitigate the unacceptable toxic side effects have involved targeting the boron atoms to tumor sites by conjugating them to site-specific antibodies. The methods of the invention can be used as an adjunct to provide a radioprotective treatment against the alpha radiation of the activated boron atoms using PTHrP. Systemic administration of the PTHrP will allow administration of the boron compound either locally at the tumor site or systemically with the PTHrP being able to prevent or reverse hematopoietic toxicity of the targeted cytotoxic neutron capture therapy.

The methods of the invention also can be used in other applications in which cytotoxic agents, particularly those that affect the lymphoid system (and therein particularly the T-lymphocytes), are used to depress host immunity in certain autoimmune diseases, e.g., systemic lupus erythematosis, and in patients receiving organ transplants. Often the cytotoxic drugs used in these disorders are similar to those often used in cancer chemotherapy, with the attendant myeloid and other hematopoietic side effects. In addition to these drugs, specific antibodies against lymphoid cells (particularly T-cells), e.g., the anti-Tac monoclonal antibody of Uchiyama et al., J. Immunol. 126:1393 and 1398 (1981), which specifically binds to the human IL-2 receptor of activated T-cells, have been conjugated to cytotoxic agents, such as drugs, toxins or radioisotopes, to effect a relatively select killing of these cells involved in organ rejection. For example, a T-cell antibody can be conjugated with a beta- or alpha-emitting radioisotope, and this can be administered to the patient prior to undertaking organ transplantation and, if needed, also thereafter. In order to effect a high T-cell killing dose without the concomitant limiting side effects to the hematopoietic system, the treatment of the T-cell killing dose of therapy can be combined with the use of PTHrP, according to the present invention. This method is useful for the long-term survival of many organ transplants, such as the kidney, heart, liver, etc., because such transplants often are accompanied by a critical period during which there is a risk of organ rejection. The PTHrP therapy can serve as an adjunct therapy for organ transplants.

The dosage level of the PTHrP will be a function of the extent of compromise of the hematopoietic cells, correlated generally with the peripheral blood cell count in the patient. Periodic monitoring of the WBC, RBC, platelets and other blood cell counts and adjustment of the rate and frequency of infusion or the dosage of the PTHrP administered to achieve a relatively constant level of peripheral blood cell count will ensure that the patient does not sustain undue marrow toxicity from the therapy. Experience will permit anticipation of when the treatment will lower the levels of the circulating blood components and infusion of the PTHrP at a time and in an amount sufficient to substantially prevent depression to the components of the blood in response to the cytotoxic therapy.

The present invention includes administration PTHrP either alone or in combination with one or more cytokines, preferably lymphokines, prior to, together with and/or subsequent to administration of cytotoxic radioisotopes, drugs and/or toxins, alone or in combination, as such or in the form of antibody conjugates. The guidelines provided herein will enable the ordinary skilled clinician to adapt PTHrP administration to enhance the efficacy and mitigate the adverse hematopoietic side effects of cytotoxic therapy as a function of WBC, platelet and erythrocyte counts, marrow component status and other particular diagnostic indicia peculiar to the individual patient. In general, this invention is applicable to support an enhancement of efficacy of any cytotoxic therapy that has serious hematopoietic side effects that limit the therapy's efficacy.

In a specific embodiment, it is contemplated that post chemo/radiation therapy, the subject may receive a bone marrow transplant. In such a method, bone marrow cells, or stem cells that have been engineered to express properties of hematopoeitic stem cells are transplanted into the individual. The cells are such that they have been maintained and expanded outside of the patient. The cells may be heterologous (from a donor e.g., a relative) or they may be autologous (from the subject itself). The cells also may be banked peripheral hematopoietic stem cells that are reintroduced into the patient post chemo/radiation therapy. In autologous bone marrow transplant (ABMT) the patient will serve as his/her own bone marrow donor. Thus, a normally lethal dose of irradiation or chemotherapeutic may be delivered to the patient to kill tumor cells, and the bone marrow repopulated with the patients own cells that have been maintained (and perhaps expanded) ex vivo. Because, bone marrow often is contaminated with tumor cells, it is desirable to purge the bone marrow of these cells, thus the cells, once harvested may be irradiated to kill the cancer cells therein and then expanded in culture ex vivo to produce an expanded population of bone marrow cells for administration to the subject. It is contemplated that in such bone marrow transplants, the PTHrP (full length PTHrP, or related PTHrP polypeptide fragment, analog, or derivative thereof) may be used to enhance engraftment/survival of bone marrow transplants and decreases the period of cytopenia and window of opportunity for opportunistic infections that are prevalent post chemo/radiation.

The components and compositions for the above methods are described in further detail below.

A. PTHrP, Analogs and Derivatives Thereof The methods and compositions of the present invention relate to the use of PTH related peptide (PTHrP) for use as a hematoprotectant in any therapy where it is desirable to prevent cytotoxic cytopenia. PTHrP was previously known as the factor responsible for humoral hypercalcemia of malignancy and is a peptide of 138-174 amino acids (depending on alternative splicing) which binds to the PTH/PTHrP receptor. The compositions of the invention may employ a full length PTHrP; a truncated

PTHrP polypeptide that is physiologically active, or a polypeptide analog of PTHrP. Such proteins or polypeptides may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art and are discussed in further detail below.

The N-terminal 34 amino acid sequence of PTHrP has limited sequence homology to that of parathyroid hormone. PTHrP is generally less potent and less bone anabolic than PTH. The sequence of hPTHrP (1-34) is as follows:

Ala VaI Ser GIu His GIn Leu Leu His Asp Lys GIy Lys Ser He GIn Asp Leu Arg Arg Arg Phe Phe Leu His His Leu He Ala GIu He His Thr Ala (SEQ ID NO:1).

Those of skill in the art have reported several truncated homologs and analogs of PTHrP. Analogs in which amino acid residues 22-31 of PTHrP(I -34) are replaced by an amphipathic α-helix (U.S. Pat. No. 5,589,452 and WO 97/07815) and related derivatives have been described as useful for treating osteoporosis. (Vickery et al. J. Bone & Mineral Research, 11(12):1943-1951 (1996); D. Leaffer et al. Endocrinology, 136(8):3624-3631 (1995)).

In addition, monocyclic and bicyclic analogs of PTHrP (1-34) and PTHrP(7-34) were shown to bind strongly to the PTH receptor and stimulate (or antagonise) PTH-stimulated adenyl cyclase activity in osteoblast-like cells. (Michael Chorev et al. Biochemistry, 36:3293- 3299 (1997), and "Cyclic analogs of PTH and PTHrP," WO 96/40193).

U.S. Patent No. 6,583,114 provides compositions of PTHrP in which amino acid residues 22-31 form an amphipathic α-helix. U.S. Patent No. 6,849,710, shows methods and compositions for synthesizing various useful analogs of PTHrP. Other PTHrP compositions that may be used in the present invention are described in e.g., U.S. Patent 6,362,163 and U.S. Patent 6,147,186, which provide descriptions of PTHrP compositions having the amino- acid sequence:

1-Ala VaI Ser GIu His GIn Leu Leu His Asp Lys GIy Lys Ser He GIn Asp Leu Arg Arg Arg Phe Phe Leu His His Leu He Ala GIu He His Thr Ala GIu Tyr -36 in which amino acid 23 or amino acids 5 and 23 are altered.

Other compositions that may be useful include analogs of PTHrP described in U.S. Patent 6,503,534; U.S. Patent 6,544,949; U.S. Patent 5,723,577; U.S. Patent 5,955,574; U.S. Patent 5,969,095; U.S. Patent 6,921,750 each of which provide a teaching of composition that comprise [Glu22,25, Leu23,28,31, Aib29, Lys26,30] PTHrP (1-34)NH₂. U.S. Patent 5,688,760 describes polypeptides comprising an N-terminal amino acid sequence corresponding to amino acids 107-111 of PTHrP. The PTHrP may be provided as a fusion protein for example as a fusion protein with an Fc region as described in U.S. Patent 6,756,480. U.S. Patent 6,472,505 and U.S. application 2002/0132973describes useful cyclic and acyclic analogs of hPTH (1-34) and hPTHrP (1-34), pharmaceutical compositions of such analogs and methods of using the same in treatment of diseases associated with calcium regulation with those analogs.

"A truncated PTHrP polypeptide that is physiologically active" is a polypeptide that has a sequence that is less than the full complement of amino acids found in full length PTHrP which, nonetheless, elicits a physiological response that is associated with PTHrP.

Such a response may be greater or smaller in magnitude than the response seen from the same concentration of full length PTHrP. The truncated PTHrP need not be fully homologous with PTHrP to elicit a similar physiological response. Typically, the truncated PTHrP will be truncated from the C-terminus and will range from 30 to 40 residues, with PTHrP(I -32), PTHrP(l-34), PTHrP(l-36), PTHrP(I -37),and PTHrP(l-38) being preferred, but not exclusive, representatives of this group. In other particular embodiments, the PTHrP may be the N-terminal sequences of between 30 to 50 residues of PTHrP, preferably from 1-32, 1-33, 1-34, 1-35, 1-36, 1-37 and 1-38, are particularly contemplated to be useful in the present invention. It should be noted that the PTHrP truncated sequences may be C-terminally truncated as compared to wild-type, N-terminally truncated as compared to wild-type or may be fragments of PTHrP that have been generated from the middle of the protein i.e., are both N- and C-terminally truncated as compared to wild-type.

A "polypeptide analog of PTHrP" refers to a polypeptide having art-accepted substitutions, deletions or insertions relative to wild-type PTHrP or is substantially homologous to PTHrP such that the analog has a similar physiological activity. An analog as used herein is thus any variant of the native wild-type PTHrP in which one or more of the amino acids has been substituted by another amino acid or an amino acid derivative. The analog may be an analog of the full-length PTHrP or alternatively, the analog will be an analog of a truncated PTHrP that polypeptide that is physiologically active. Those of skill in the art are referred to e.g., U.S. Patent 5,874,086; U.S. Patent 5,798,225; U.S. Patent

5,807,823; U.S. Patent 5,821,225; U.S. Patent 5,977,070; U.S. Patent 6,051,686; U.S. Patent 5,589,452; U.S. Patent 5,693,616; U.S. Patent 5,695,955; U.S. Patent 5,840,837; U.S. Patent 6,147,186; U.S. Patent 6,362,163; U.S. Patent 6,537,965; U.S. Patent 6,495,662; RE37919 Reissue of U.S. Patent 5,171,670; U.S. Patent 6,417,333; U.S. Patent 6,803,213; U.S. Patent 5,886,148; U.S. Patent 5,955,425; U.S. Patent 6,541,450; U.S. Patent 6,316,410; U.S. Patent 5,556,940; U.S. Patent 6,503,534; U.S. Patent 6,544,949; U.S. Patent 5,723,577; U.S. Patent 5,955,574; U.S. Patent 5,969,095; U.S. Patent 6,921,750; U.S. Patent 6,583,1 14; U.S. Patent 6,849,710; U.S. Patent 6,787,518 and U.S. applications 2003/0144209 2003/0203012; and 2005/0272660 which provide teachings of exemplary such analogs. Each of the foregoing patents, along with the other patents discussed above are expressly incorporated herein by reference as providing teachings of methods and composition for preparing PTHrP and PTHrP analogs that may be useful in the methods of the present invention. A particularly useful analog for use in the methods of the present invention is

Semparatide™. Semparatide is a synthetic analog of naturally occurring PTHrP (1-34). Semparatide comprised of a linear chain of 34 natural amino acids, and differs from PTHrP in the substitution of residues 22-31 with residues that form an amphiphilic helix. The sequence of the molecule is:

SEQ ID NO 1: A-V-S-E-H-Q-L-L-H-D-K-G-K-S-I-Q-D-L-R-R-R-E-L-L-E-K-L-L-E-K-L- H-T-A

The compound may be produced by recombinant means or may be synthesized by solid- phase polymer supported synthesis, using Fmoc-compatible coupling techniques, with acid labile side chain protection. Following completion of synthesis and deprotection, the compound may be cleaved from the solid support, purified by RP-HPLC and salt exchange (to the X acetate), and lyophilized (to afford the X acetate, N hydrate). The acetate is freely soluble in water. This compound has the formula:

H-Ala-Val^er-G-u-His-Gln-Leu-Leu-His-Asp-

1 2 3 4 5 6 7 8 9 10

Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg- t l 12 13 14 15 16 17 18 19 20

Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Glu-Lys-

21 22 23 24 25 26 27 23 29 30

Leu-Hk-Thr-Aia.NH2 ^• JCC2H4O2 ^• >«2θ 31 32 33 34

While it is preferred that the PTHrP full length proteins, PTHrP truncated proteins that are physiologically active, or the PTHrP analogs are derived from a native sequence that is a human sequence, it should be understood that such PTHrP compositions may be PTHrP compositions of any mammalian species, e.g., human, bovine, porcine or rabbit may be used in this invention, with human PTHrP being the preferred source. One of skill in the art will recognize that substitution, deletion and insertion variants of the preferred embodiments enumerated below, according to the art-accepted principles described above, are also within the scope of the invention.

Typically, the dosage of the PTHrP or analogue or derivative thereof will range between about 0.01 and 10 μg/kg body weight per day, preferably from about 0.1 to about 0.5 fg/kg body weight per day. For a 50 kg human subject, the daily dose of active ingredient is from about 0.5 to about 100 μgs, preferably from about 5 to about 10 μgs. This dosage may be delivered in a conventional pharmaceutical composition by a single administration, by multiple applications, or via controlled release, as needed to achieve the most effective results. Dosing will continue for as long as is medically indicated, and may range from a few weeks to several months. Such dosing may be intermittent or it may be daily. Preferably the dosage is initiated prior to administration of the cytotoxic therapy and may continue during and after the cytotoxic therapy has stopped.

The invention also contemplates the use of PTHrP derivatives. As used herein the term PTHrP derivative is used to denote a PTHrP molecule that has been altered to modify properties other than its physiological properties. For example, the PTHrP molecule may be derivatized to make it more accessible. It may be derivatized to have added to it an agent that increases its uptake or its targeting. For example attaching the PTHrP to a diphosphonate or orthophosphate will facilitate the targeting of the PTHrP (or related polypeptide fragment, analogue or derivative thereof) to the bone marrow as will attaching it to certain antibodies or Fc regions of certain antibodies. The PTHrP may be derivatized with a water-soluble polymer. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers, and modify the rate of clearance from the body. (Greenwald et al., Crit Rev Therap Drug Carrier Syst. 2000; 17: 101-161; Kopecek et al., J Controlled Release., 74:147-158, 2001). To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

Polyethylene glycol (PEG), has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification (Harris et al., Clin Pharmacokinet. 2001;40(7):539-51). Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. (Greenwald et al., Crit Rev Therap Drug Carrier Syst. 2000;17:101-161; Zalipsky et al., Bioconjug Chem. 1997;8:11 1-118). PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications (Nathan et al., Macromolecules. 1992;25:4476-4484; Nathan et al., . Bioconj Chem. 1993;4:54-62).

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consists of alternating polymers of PEG and tri-functional monomers such as lysine have been used by VectraMed (Plainsboro, NJ). The PEG chains (typically 2000 daltons or less) are linked to the a- and e- amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 45 kDa). Thus, PEGylated proteins in the range of between 20 and 35 kDa in molecular weight will be useful. In addition, to the polymer backbone being important in maintaining circulatory half- life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease- specific enzymes (see e.g., technologies of established by VectraMed, Plainsboro, NJ). Such linkers may be used in modifying the PTHrP proteins described herein for therapeutic delivery.

B. Preparation and Purification of PTHrP, Analogs and Derivatives Thereof The PTHrP proteins or fragments thereof can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., (1984);Tam et al., J. Am. Chem. Soc, 105:6442, (1983); Merrifield, Science, 232: 341-347, (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic

Press, New York, 1-284, (1979), each incorporated herein by reference. The proteins can be readily synthesized and then screened in PTHrP receptor binding/activity assays to determine whether the proteins produced possess the requisite PTHrP-like activity as an initial screen. The proteins/fragments also may be tested in an exemplary physiological assays. Any PTHrP-derived protein that has at least some physiological effect associated with PTHrP may be used in the methods of the present invention.

As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides and a variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. Recombinant expression of proteins is routine and well known to those of skill in the art. Expression may be achieved in microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., Biotechnol Appl Biochem., 30 ( Pt 3):235-44, 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., MoI Ther. 6(1):5-1 1, 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., Biotechnol Genet Eng Rev.; 17:213-52, 2000); or animal cell systems. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art.

Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), Wl 38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC 12, K562 and 293 cells. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, MoI Biotechnol. 16(2):151-60, 2000; Colosimo et al., Biotechniques, 29(2):314-8, 2000. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post- translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

In the recombinant production of proteins of the invention, it would be necessary to employ vectors comprising polynucleotide molecules for encoding the PTHrP-derived proteins. Methods of preparing such vectors as well as producing host cells transformed with such vectors are well known to those skill in the art. Recombinant expression may employ a selectable marker and an origin of replication, for propagation in a host. These elements of the expression constructs are well known to those of skill in the art. Generally, the expression vectors include DNA encoding the given protein being operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation. The terms "expression vector," "expression construct " or "expression cassette " are used interchangeably throughout this specification and are meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The choice of a suitable expression vector for expression of the PTHrP peptides or polypeptides of the invention will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan. Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (MoI. Cell. Biol. 3:280 (1983)); Cosman et al. (MoI. Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982. Other considerations for producing expression vectors are detailed in e.g., Makrides et al., Protein Expr Purif, 17(2): 183-202; 1999; Kost et al., Curr Opin Biotechnol., 10(5):428-33, 1999. Wurm et al., Curr Opin Biotechnol. 10(2): 156-9, 1999 is incorporated herein as teaching factors for consideration in the large- scale transient expression in mammalian cells for recombinant protein production. The expression construct may further comprise a selectable marker that allows for the detection of the expression of a peptide or polypeptide. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin, DHFR, zeocin and histidinol. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic), β-galactosidase, luciferase, or chloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed. Immunologic markers also can be employed. For example, epitope tags such as the FLAG system (IBI, New Haven, CT), HA and the 6xHis system (Qiagen, Chatsworth, CA) may be employed. Additionally, glutathione S-transferase (GST) system (Pharmacia, Piscataway, NJ), or the maltose binding protein system (NEB, Beverley, MA) also may be used. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.

Expression requires that appropriate signals be provided in the vectors, such as enhancers/promoters from both viral and mammalian sources that may be used to drive expression of the nucleic acids of interest in host cells. Usually, the nucleic acid being expressed is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the protein of interest (e.g., the PTHrP or any of the hematopoietic factors discussed herein, variants thereof and the like). Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence if the promoter nucleotide sequence directs the transcription of the sequence.

Similarly, the phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. Any promoter that will drive the expression of the nucleic acid may be used. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter. Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, β-actin, rat insulin promoter, the phosphoglycerol kinase promoter and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient to produce a recoverable yield of protein of interest. By employing a promoter with well known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Inducible promoters also may be used.

Another regulatory element that is used in protein expression is an enhancer. These are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Where an expression construct employs a cDNA insert, one will typically desire to include a polyadenylation signal sequence to effect proper polyadenylation of the gene transcript. Any polyadenylation signal sequence recognized by cells of the selected transgenic animal species is suitable for the practice of the invention, such as human or bovine growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences. The termination region which is employed primarily will be one selected for convenience, since termination regions for the applications such as those contemplated by the present invention appear to be relatively interchangeable. The termination region may be native with the transcriptional initiation, may be native to the DNA sequence of interest, or may be derived for another source.

Further, it has been shown that polyhistidylation of nucleic acid molecules is useful in achieving cytosolic delivery of nucleic acids, and that ionic complexes between histidylated polylysine and a pDNA are attractive for developing a nonviral gene delivery system (Midoux et al., Somat Cell MoI Genet. 2002 Nov;27(l-6):27-47).

Site-specific mutagenesis may be useful in the preparation of individual PTHrP related proteins used in the methods of the invention. This technique employs specific mutagenesis of the underlying DNA (that encodes the amino acid sequence that is targeted for modification). The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids also are routinely employed in site-directed mutagenesis, and eliminates the step of transferring the gene of interest from a phage to a plasmid. In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization (annealing) conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation- bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non- mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

Of course, the above described approach for site-directed mutagenesis is not the only method of generating potentially useful mutant peptide species and as such is not meant to be limiting. The present invention also contemplates other methods of achieving mutagenesis such as for example, treating the recombinant vectors carrying the gene of interest mutagenic agents, such as hydroxylamine, to obtain sequence variants.

It will be desirable to purify the peptides of the present invention. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the peptides or polypeptides of the invention from other proteins, the polypeptides or peptides of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide include size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, isoelectric focusing and capillary electrophoresis. A particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC). Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded polypeptide, protein or peptide. The term "purified polypeptide, protein or peptide" as used herein, is intended to refer to a composition, isolated from other components, wherein the polypeptide, protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified polypeptide, protein or peptide therefore also refers to a polypeptide, protein or peptide, free from the cellular environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide, protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the polypeptide, protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide, protein or peptide.

C. Combination Therapy with other Hematoprotectants

Hematopoietic reconstitution has previously been achieved by administration of cytokine growth factors such as Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13; IL-15, IL- 16, IL- 17, stem cell factor (also known as c-kit ligand), and M-CSF, or PEGylated derivatives thereof, as soon as possible after the subject has been exposed to the chemotherapeutic and/or radiotherapeutic agent. In the present invention, in order to combat the myelosuppression observed with the chemotherapy the PTHrP-based methods may be used in combination with cytokine therapy. Combination therapy contemplated herein includes administration of one or more hematopoietic factors selected from the group consisting of Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13; IL-15, IL-16, IL-17, stem cell factor (also known as c-kit ligand), and M-CSF, or PEGylated derivatives thereof in combinations with PTHrP (the hematopoietic factor may be administered either concurrently with PTHrP, or prior to, and preferably subsequent to the PTHrP) is particularly contemplated. Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-13; IL-15, IL-16, IL-17, stem cell factor (also known as c-kit ligand), and M-CSF, are merely exemplary suitable immunomodulators that can be used in combination with PTHrP or an analogue thereof for adjunct therapy. U.S. patent No. 5,120,525, which is incorporated by reference, provides methods of use of such agents in adjunct immunomodulator therapy.

Cytokines, or growth factors, are hormone-like peptides produced by diverse cells and are capable of modulating the proliferation, maturation and functional activation of particular cell types. Herein, cytokines refer to a diverse array of growth factors, such as hematopoietic cell growth factors (e.g., erythropoietin, colony stimulating factors and interleukins), nervous system growth factors (e.g., glial growth factor and nerve growth factor), mostly mesenchymal growth factors (e.g., epidermal growth factor), platelet-derived growth factor, and fibroblast growth factor I, II and III and the like. Several cytokines may act in concert to induce cell differentiation and maturation, and such cytokines may have other biological functions. As such, limiting the amount of cytokine to be used in treating a patient that has undergone myelosuppressive therapy is desirable. The combined use of PTHrP with the cytokine-based therapy will allow the use of less cytokine for treating the subject post radiation or post chemotherapy than the amount that would be needed in the absence of the PTHrP.

In the case of IL-I, there may be several forms, such as IL-I -alpha and IL-I -beta, which nevertheless appear to have a similar spectrum of biological activity. Preferred cytokines for use in the method and compositions of the invention are lymphokines, i.e., those cytokines which are primarily associated with induction of cell differentiation and maturation of myeloid and possibly other hematopoietic cells. A preferred lymphokine is IL- 1. Other such lymphokines include, but are not limited to, G-CSF, M-CSF, GM-CSF, Multi- CSF (IL-3), and IL-2 (T-cell growth factor, TCGF). IL-I appears to have its effect mostly on myeloid cells, IL-2 affects mostly T-cells, IL-3 affects mutiple precursor lymphocytes, G- CSF affects mostly granulocytes and myeloid cells, M-CSF affects mostly macrophage cells, GM-CSF affects both granulocytes and macrophage. Other growth factors affect immature platelet (thrombocyte) cells, erythroid cells, and the like. The cytokines may be used alone or may be provided in a combination of two or more cytokines in order to provide protection against, mitigation and/or reversal of myeloid or hematopoietic toxicity associated with cytotoxic agents when administered in combination with the PTHrP as described herein. Examples of possible combinations include IL-I + GC- CSF + PTHrP, IL-I + IL-3 + PTHrP, G-CSF + IL-3 + PTHrP, IL-I + platelet growth factor + PTHrP and the like. Certain combinations will be preferred, depending on the maturation state of the target cells to be affected, and the time in the course of cytotoxic action that the protective agent needs to be administered. For example, in patients with depression of several hematopoietic cell types (e.g., myeloid, lymphoid and platelet), a combination of IL-I + IL-3/and/or platelet growth factor in combination with PTHrP may be particularly useful, while more severe depression of the myeloid series may require such combinations as IL-I + G-CSF in combination with the PTHrP. Certain cytotoxic agents have greater compromising effects on particular hematopoietic elements, either because of the nature of the agent or the dosage necessary to achieve a therapeutic effect, and the appropriate choice, dosage and mode of administration of cytokine(s) will follow from such effects.

In addition to the cytokines, the PTHrP may be provided in combination with other compounds or techniques for preventing, mitigating or reversing the side effects of cytotoxic agents. Examples of such combinations include, e.g., administration of the PTHrP and a cytokine together with a second antibody for rapid clearance, as described, e.g., in Goldenberg, U.S. Patent 4,624,846, from 3 to 72 hours after administration of a targeted primary antibody or antibody fragment conjugate (with a radioisotope, drug or toxin as the cytotoxic component of the immunoconjugate) or of a non-conjugated drug or toxin, to enhance clearance of the conjugate, drug or toxin from the circulation and to mitigate or reverse myeloid and other hematopoietic toxicity caused by the therapeutic agent. In all aspects of the invention, it is a desired goal bring the peripheral blood cell counts and differential to within the normal levels or as close thereto as possible. The RBC level in terms of hemoglobin concentration, which is normally 13.5-17.5 gm/dL in men and 1 1.8 - 15.5 gm/dL in women, is reduced to less than 10 gm/dL with chemotherapy or radiotherapy; WBC count which is normally in the range of 4-1 lxlO⁹/L is reduced to less than lxlO⁹/L in subjects in response to chemotherapy of radiotherapy; platelets are in a normal range of 150,000 to 400,000/μL but are reduced to 50,000/uL in response to chemotherapy or radiotherapy. The methods of the invention are useful in that where the cytotoxic therapy produces such a reduction in blood cell counts, this reduction can be reversed by administration of either PTHrP alone or a combination of PTHrP with one or more cytokines and or one or more other agents. In embodiments where cytokine is administered in combination with the PTHrP to return the blood component levels to be close to normal. It is contemplated that the cytokine is administered in a dose of from about 1 μg to about 500 μg, preferably 5-100 μg, more preferably about 10 ug. The clinician will appreciate that variations in the timing and dosage of cytokine administration as a function of the type of cytokine used, the extent and rate of compromise of the bone marrow and/or other components of the myeloid and/or other hematopoietic elements and the individual characteristics of the patient and the therapy protocol will be possible and often desirable. These can easily be made by the clinician using conventional monitoring techniques and dosimetric principles.

D. Normal Levels of Blood Components

Throughout the methods of the present invention, it will be desirable to monitor changes in the subjects blood cell populations. The complete blood cell count will routinely be monitored prior to initiation of the therapeutic regimen as well as during and post-therapy follow-up. During treatment and follow-up, monitoring the patient's blood cell count is important (e.g., prior to each treatment cycle and 10-14 days after each treatment cycle). A complete blood count provides important information about the kinds and numbers of cells in the blood, including red blood cells, white blood cells, and platelets. A complete blood count typically will involve determining a white blood cell

(WBC) count. These cells protect the body against infection and the number of white cells can increase dramatically upon infection or chemo- or radio-therapeutic insult, as such, the number of white blood cells is sometimes used to identify an infection or monitor the body's response to cancer treatment. Analysis of the white blood cell types (WBC differential) in blood will be a useful indicator for the efficacy of the methods of the invention. The major types of white blood cells are neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Immature neutrophils, called band neutrophils, are also included and counted as part of this test. Each type of cell plays a different role in protecting the body. The numbers of each one of these types of white blood cells give important information about the immune system. An increase or decrease in the numbers of the different types of white blood cells can help identify infection, an allergic or toxic reaction to certain medications or chemicals, and many conditions, such as leukemia. To assess white blood cell (WBC) count for the present invention, a health professional will look at both the number (WBC count) and the WBC differential. To determine whether there are too many or too few of a certain type of cell, the total count is multiplied by the percentage of that particular cell. There are normal values for the total number of each type of white cell

The typical normal range of white blood cells in males and non-pregnant females is between 4,500-11,000 cells/microliter (μL)³ or 4.5-11.0 x 10⁹/Liter (SI units). It is contemplated that the PTHrP compositions administered in accordance with the present invention will maintain or return the subjects white blood cell count to within this normal range. The average distribution of white blood cell types 47%-77% neutrophils; 0%-3% band neutrophils; 16%-43% lymphocytes, 0.5%-10%, monocytes, 0.3%-7% eosinophils, and 0.3%-2% basophils. In further embodiments, the PTHrP administration in accordance with the methods of the present invention will balance the white blood cell differential such that the white blood cell components are present in these appropriate distributions relative to each other.

In addition to white blood cell counts, it may be desirable to obtain a red blood cell count (average value 4.6-6.2 million RBCs per microliter (μL) or 4.6-6.2 x 10^l2/Liter (SI units), a hematocrit (40%-54% in men; 37%-47% women; 31-41% children; 44%-64% in newborns) and/or perform hemoglobin test (14-18 g/dL or 8.7-11.2 mmol/L (SI units) in men and 12-16 g/dL or 7.4-9.9 mmol/L in women). Other factors that can be monitored include hemoglobin levels, erythrocyte sedimentation rate (ESR), erticulocyte count, red eel distribution width, and red blood cell indices.

The platelet count also could be determined. The average normal platelet count is 140,000-450,000 platelets per mm³ or 150-400 x 109/Liter (SI units). Methods for performing such tests for various blood components are known to those of skill in the art.

£. Pharmaceutical Compositions

Pharmaceutical compositions for administration according to the present invention can comprise at least one PTHrP-derived protein (e.g., a full length PTHrP polypeptide, or a variant or analog thereof or any other PTHrP-related protein that protects hematopoeitic cells from the cytotoxic effects of chemo- or radiotherapy). The pharmaceutical compositions also may include other therapeutic agents. For example, while the present invention stems from the unique discovery of the therapeutic efficacy of PTHrP compositions in the protection of hematopoietic cells from cytotoxic chemotherapy or radiotherapy, it is contemplated that the PTHrP may be administered not only alone but in combination with other therapeutic regimens designed for the treatment of cytotoxic agent-induced cytopenia (e.g., anemia, neutropenia and thrombocytopenia). Any increase in the level of peripheral blood cell count or any component of the blood count in response to such therapy will be a useful therapeutic outcome. As discussed above, cytokines such as Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL- 12, IL-13, IL- 15, IL- 16, IL- 17, stem cell factor (also known as c-kit ligand), and M-CSF, and the like may be useful in a combination therapy protocol. As such, one or more of cytokines may be provided in the pharmaceutical compositions to be used in the treatment of subjects that are about to undergo chemotherapy, radiotherapy or some other therapy that will have a myelosuppressive effect

Each of the therapeutic preparations is preferably provided in a pharmaceutically acceptable form optionally combined with a pharmaceutically acceptable carrier. These compositions can be administered by any means that achieve their intended purposes. Individualized amounts and regimens for the administration of the compositions for the treatment of infertility using the methods of the present invention can be determined readily by those with ordinary skill in the art using the guidance provided herein to determine the efficacy of a dosage in an animal model and then to increase the dosage to higher mammals. Administration of many cytokines are known to those of skill in the art and can readily be found in the Regimens for use of PTH in other indications also are known and discussed in the Physician's Desk Reference. That document provides exemplary guidance as to types of formulations, routes of administration and treatment regimens that may be used in administering PTH and other hormones. Such routes teachings can be used for the administration of PTHrP. Any of the protocols, formulations, routes of administration and the like described therein can readily be modified for use in the present invention. Compositions within the scope of this invention include all compositions comprising at least one PTHrP-related polypeptide formulated in an amount effective to achieve its intended purpose of protecting a composition of hematopoietic cells from cell death and/or preventing or overcoming myelosuppression. The active agents used in the methods of the present invention may be administered by any means normally employed for such administration.

It is understood that the suitable dose of a composition according to the present invention will depend upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

However, the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This typically involves adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight. Peripheral blood cell counts and differentials will be a useful determinant in assisting the clinician to determine and/or adjust the dose.

The total dose required for each treatment may be administered in multiple doses or in a single dose. The compositions may be administered alone or in conjunction with other therapeutics directed to the disease or directed to other symptoms thereof.

The compositions of the invention should be formulated into suitable pharmaceutical compositions, i.e., in a form appropriate for in vivo applications in a the therapeutic intervention of myelosuppression. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals, preferably for oral administration. The hormone formulations may be formulated akin to the currently available hormonal preparations. The peptide/protein formulations may be formulated similarly to any other small protein composition. All routes of administration are contemplated (e.g. Such routes of administration include, subcutaneous, intravenous, intradermal, intraaterial, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term release), aerosol, sublingual, nasal, anal, vaginal, oral, or transdermal delivery, or by surgical implanation). Transdermal patches also may be used. Intranasal administration is of particular interest.

One will generally desire to employ appropriate salts and buffers to render the compositions stable and allow for uptake of the compositions at the target site. Generally the protein compositions of the invention are provided in lyophilized form to be reconstituted prior to administration. Buffers and solutions for the reconstitution of the compositions may be provided along with the pharmaceutical formulation to produce aqueous compositions of the present invention for administration. Such aqueous compositions will comprise an effective amount of each of the therapeutic agents being used, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the therapeutic compositions, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

Methods of formulating proteins and peptides for therapeutic administration also are known to those of skill in the art. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Conventional routes of administration include, subcutaneous, intravenous, intradermal, intraarterial, intramusclar, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term release), aerosol, sublingual, nasal, anal, vaginal, oral, or transdermal delivery, or by surgical implantation at a particular site. The treatment may consist of a single dose or a plurality of doses over a period of time. Preferably, the route of administration is chosen for the therapeutic intervention to have maximum impact on the circulating blood cell counts.

The active compounds may be prepared for administration as solutions of free base or pharmacologically acceptable salts in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. The compositions of the present invention may be formulated in a neutral or salt form.

Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

"Unit dose" is defined as a discrete amount of a therapeutic composition dispersed in a suitable carrier. Parenteral administration of the therapeutic compounds may be carried out with an initial bolus followed by continuous infusion to maintain therapeutic circulating levels of drug product. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient.

The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the routes of administration. The optimal pharmaceutical formulation will be determined by one of skill in the art depending on the route of administration and the desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface areas or organ size. Further refinement of the calculations necessary to determine the appropriate treatment dose is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein as well as the pharmacokinetic data observed in animals or human clinical trials.

Appropriate dosages may be ascertained through the use of established assays for determining blood levels in conjunction with relevant dose response data. The final dosage regimen will be determined by the attending physician, considering factors which modify the action of drugs, e.g., the drug's specific activity, severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding appropriate dosage levels and duration of treatment for specific diseases and conditions. It will be appreciated that the pharmaceutical compositions and treatment methods of the invention may be useful in fields of human medicine and veterinary medicine. Thus the subject to be treated may be a mammal, preferably human or other animal. For veterinary purposes, subjects include for example, farm animals including cows, sheep, pigs, horses and goats, companion animals such as dogs and cats, exotic and/or zoo animals, laboratory animals including mice rats, rabbits, guinea pigs and hamsters; and poultry such as chickens, turkeys, ducks and geese.

The present invention also contemplated kits for use in the treatment of myelosuppression. Such kits include at least a first composition comprising the PTHrP-based proteins/peptides described above in a pharmaceutically acceptable carrier. Another component may be an agent used in treating myelosuppression (e.g., a composition that contains one or more cytokines etc.) in a pharmaceutically acceptable carrier. A third component may be the agent that is used for the radiotherapy or chemotherapy. The kits may additionally comprise solutions or buffers for effecting the delivery of the first, second and third compositions. The kits may further comprise catheters, syringes or other delivering devices for the delivery of one or more of the compositions used in the methods of the invention. The kits may further comprise instructions containing administration protocols for the therapeutic regimens.

Example 1 — Mouse model

14 days of pretreatment with a daily dose of semparatide enlarges the granulocyte and lymphoid compartments in mice. These cells are available for release following a challenge with a single dose of 5-fluorouracil and can be quantified by counting cells in the systemic circulation during the two weeks post chemotherapy. Semparatide induces an increase in the area under the curve of circulating white blood cells, lymphocytes and neutrophils post chemotherapy. These enhanced levels of circulating white blood cells, lymphocytes and neutrophils would be expected to improve survival in animals that would otherwise succumb to opportunistic infections during the post chemotherapeutic or post irradiation period of myelosupression. Results of pretreatment with semparatide in mice treated with 5- fluorouracil are shown in Figures 1-3. These results show that pretreatment with semparatide at a daily dose of 200/mg/kg for 14 days prior to the induction of myelosupression by a single dose of 225mg/kg of 5-fluorouracil at day 0 reduces the cytoreduction of granulocytes and lymphocytes immediately following therapy and stimulates a significantly greater cellular response within 14 days after cytoreduction. These results are significantly better that those observed in this model even after 14 days of twice daily injections of G-CSF.

Example 2 - Clinical Trials

A patient that is about to undergo chemotherapy or radiation therapy for cancer is selected for treatment. The patient is treated once daily with 50 μg PTHrP or an analogue by subcutaneous injection (on each days -7 to day 0).

On day 0, the patient is treated with chemotherapy or radiation therapy. Twenty four hours after administration of the chemotherapy (day 1) and for the next 13 days (days 2-14) the patients will receive a once daily subcutaneous injection of PTHrP or analogue at the same pre-chemotherapy dose.

In chemotherapy or radiotherapy, there is often a drop in peripheral blood cell count. In this case, as a result of the treatment with the 50 ug PTHrP or analogue thereof, it is found that the usual drop in peripheral blood cell count is inhibited significantly, thus permitting the patient to receive more extensive chemotherapy or radiotherapy.

Frequent blood sampling will allow the physician to terminate PTHrP therapy prior to day 14 if circulating levels of RBCs, WBCs or platelets return to pre-treatment (normal) levels. As a result of administration of the PTHrP in combination with the chemotherapy or radiotherapy, the patient is able to tolerate greater amounts of the chemotherapy or radiotherapy and as such the therapeutic efficacy of the chemotherapy or radiotherapy is enhanced.

Simply, by way of example, and without intending to limit the invention, in some embodiments, the patient to be treated is one that has metastatic colon cancer. In exemplary treatment protocols such a patient is treated with 5-fluorouracil (5-FU) as the chemotherapeutic agent. Seven days prior to initiation of the 5-FU therapy 50 μg PTHrP or an analogue are administered to the patient by subcutaneous injection (on each days -7 to day 0). The patient is given 5-FU therapy over 5 days at a dose of 10 mg/kg/day intravenously (days 0, 1,2,3,4,5), repeated every second day during the next five days (day 7, day 9), and then twice monthly (1st and 15th day of each month) thereafter. In this type of therapy, toxicity usually begins to show by the 23rd day, whereby a drop in peripheral blood cell count. Throughout the therapy period, 50 μg PTHrP or an analogue is administered per day by subcutaneous injection. This therapy is accompanied by frequent blood sampling and the PTHrP therapy is terminated or reduced if circulating levels of RBCs, WBCs or platelets return to pre-treatment (normal) levels. As a result of this combination therapy, a significant reduction in the liver metastasis in the patient is noted on CAT scans several months after initiation of therapy.

In another example, the patient to be treated has breast cancer. Seven days prior to instituting this chemotherapy, she is started on a PTHrP or a PTHrP regimen in which she is given a dose of 50 μg PTHrP or an analogue thereof per day for seven day. On day 8 and she is to be administered adriamycin (30 mg/kg i.v. daily for 3 weeks) and cytoxin (10 mg/kg i.v. every week). In a typical treatment for breast cancer the dose of adriamycin and cytoxin has be reduced due to cytotoxicity of the drugs. In the present case, however, as a result of the prophylactic administration of the PTHrP or analogue thereof, there is marginal or low drop in her peripheral blood cell count (e.g., there is only a 10-20% drop in her WBC count). Therefore, instead of subsequent courses of chemotherapy being reduced, as is conventionally practiced when hematologic side effects occur, a full course of both drugs is repeated under continued administration of PTHrP or analogue thereof. Resolution of some of the metastatic lesions is seen radiologically several months after the initiation of the chemotherapy.

In another example, a patient presenting a peritoneal spread of a colon cancer that is non-responsive to 5-FU treatment is to be treated with radiotherapy. The patient will be treated with a 35 mCi dose of Yttriwn-90 conjugated to a F(ab')₂ fragment of a murine monoclonal antibody against carcinoembryonic antigen (CEA), by intraperitoneal injection. Seven days prior to initiating the therapy with the Yttrium-90, the patient begins receiving a daily dose of 50μg/day PTHrP or an analogue thereof (day-7 to day 0). On day 0 the patient is given the 35 mCi dose of Yttrium-90. The patient is administered PTHrP or analogue thereof at a dose of 50 μg/day over the following fourteen days. On day 2, and periodically over the next three weeks, the patient's peripheral blood cell count is taken. As long as there is no significant drop in the patient's blood cell count as compared to his normal peripheral blood cell count, the radioimmunotherapy is repeated 3 weeks later. The radioimmunotherapy may be accompanied by further administration of the PTHrP or analogue thereof. The patient is again monitored over the coming months. A further course of treatment is given 2-3 months after the initial treatment, again with administration of PTHrP or analogue thereof initiated 7 days prior to administration of the radiotherapy. Radiological evidence of some tumor and ascites reduction is monitored. Administration of the PTHrP or analogue thereof will allow the patient to tolerate higher and more frequent doses of the radioimmunotherapy.

In another exemplary therapy, the patient presents with ovarian cancer manifesting as ascites and considerable distension of her abdomen. The patient is initiated on a prophylactic course of PTHrP or analogue thereof seven days prior (days -7 to 0) to the initiation of the cancer therapy. She is given 50 μg of the PTHrP or analogue thereof per day. At day 0, is given a course of 5 FU therapy as described above. The patient continues to receive PTHrP or analogue thereof over the next 14 days. It is anticipated that the treatment with the PTHrP or analogue thereof allows the patient to tolerate multiple doses of the 5 -FU and within months of the treatment there is a reduction in the abdominal distension.

In another example, the patient to be treated is one that is to receive a kidney transplant. 7 consecutive days prior to surgery, the patient is given 50 μg/day PTHrP or analogue thereof. On the day of the surgery, the patient is given an intravenous infusion of 20 mCi Y-90 that is conjugated to an agent that targets the Y-90 to activated T-lymphocytes, in order to kill the T-cells responsible to transplant rejection. The patient is given 50 μg/day PTHrP or analogue thereof and may also receive conventional corticosteroid therapy to prevent transplant rejection. Ten days later, a second dose of 10 mCi Y-90 is given i.v. During the interim period, the patient has continued to receive 50 μg/day doses of PTHrP or analogue thereof. The patient is monitored for graft rejection and peripheral blood cell count.

In each of the above example, a 50 μg/day dose of PTHrP or analogue thereof is given. It should be understood that this is merely an exemplary dose and the dose may be varied to be more or less than that amount. In addition, while it may be convenient to maintain the same dose throughout the regimen, it may be desirable to increase or decrease the dose on any given day.

In addition the above exemplary regimens teach administration of the PTHrP or analogue thereof as a prophylactic treatment for seven days prior to the cytotoxic therapy. This seven day period is merely an exemplary treatment period and those skilled in the art will understand that the therapy may be administered for longer (e.g., 14 days or more) or shorter time periods. Moreover, while it is contemplated that the PTHrP or analogue thereof is given on consecutive days, the therapy could, if desired by given intermittently (e.g., every alternate day.) Furthermore, while a prophylactic treatment with the PTHrP or analogue thereof is contemplated, the skilled artisan will understand that the PTHrP or PTHrP analogue administration may be initiated at the same time as the cytotoxic agent therapy and need not precede the cytotoxic agent-based therapy.

As noted elsewhere, the PTHrP or analogue thereof may be administered in combination with one or more other hematopoeitic factors such as Erythropoietin, G-CSF (filgrastim), peg-G-CSF (pegfilarastim), GM-CSF (molgramostim or sargramostim), M-CSF, TCGF, flt-3, thrombopoietin, thymic stromallymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL- 12, IL-13; IL-15, IL-16, IL-17, and stem cell factor (also known as c-kit ligand). A Phase I/II trial design as using separatide as an adjunct hematoprotective agent with

Neulasta^® (pegfilgrastim) is as follows:

PHASE I/II: Safety and Proof of concept

11. Ambulatory and able to return to the site of the investigation at the specified times

Exclusion Subjects who meet any of the following exclusion criteria prior to Criteria enrollment are not eligible to participate in the study:

1. Participation in another clinical research study within 30 days prior to consenting for this study

2. Pregnant or nursing

3. Prior treatment with chemotherapy.

4. Prior radiation therapy completed < 4 weeks prior to enrollment

5. Any "currently active" second malignancy, other than non- melanoma skin cancer. Patients are not considered to have a "currently active" malignancy, if they have completed therapy and are considered by their physician to be at least less than 30% risk of relapse over next 3 months.

6. Systolic blood pressure >160 mraHg measured on at least 2 occasions.

7. HIV

8. NYHA Class III or IV Congestive Heart Failure.

9. Myocardial infarction within the 6 months prior to the first dose of study drug.

10. Serious intercurrent infections or nonmalignant medical illnesses that are uncontrolled.

11. Active psychiatric illnesses/social situations that would limit compliance with protocol requirements.

12. Any physical or mental condition in the opinion of the investigator may interfere with the subject's ability to comply with the study procedures

Study Duration 9 month recruitment period

Enrollment duration is ~3 months. Treatment with semparatide begins 7 days prior to each chemotherapy cycle.

PHASE II: Efficacy

Claims

CLAIMSWhat is Claimed Is:

1. A method of decreasing chemotherapy and/or radiation therapy- induced cytopenia in a subject comprising administering to said subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in an amount effective to decrease cytopenia in said subject as compared to the cytopenia induced in the absence of said administration.

2. The method of claim 1, wherein said administration of said composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is carried out prior to, after or subsequent to said chemotherapy and/or radiation therapy.

3. The method of claim 2, wherein said administration of said composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is carried out prior to said chemotherapy and/or radiation therapy.

4. The method of claim 3, wherein said composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered to said subject between 4-7 days prior to initiation of cytotoxic chemotherapy and/or radiation therapy.

5. The method of claim 1, wherein said cytopenia is selected from the group consisting of anemia, neutropenia and thrombocytopenia.

6. A method of decreasing myelosuppression in a subject comprising administering to said subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof.

7. The method of claim 6, wherein said myelosuppression is produced by chemotherapy or radiation therapy.

8. The method of claim 6, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered prior to chemotherapy or said radiation therapy.

9. A method of decreasing the dose or duration of chemotherapy or radiation therapy to be administered to a subject in need thereof comprising administering to said subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof.

10. A method of preventing or decreasing the level of reduction in circulating blood cells in a subject receiving radiation therapy and/or chemotherapy, comprising administering to said subject a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof prior to administration of said radiation therapy and/or chemotherapy, wherein administration of said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof prevents or produces a lower level of reduction of circulating blood cells in said subject as compared to a subject that does not receive said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof.

11. A method of treating a subject that is at risk of having a decrease in the number of cells of the hematopoietic system, comprising the step of administration to said subject an effective amount of PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered before, after, or during said chemotherapy and/or radiation therapy.

12. The method of claim 11, wherein said cells of the hematopoietic system are selected from the group consisting of, including but not limited to T cells, B cells, granulocytes, macrophages, mesenchymal cells, osteoblasts, osteoclasts and multipotent adult progenitor cells, or combinations thereof.

13. The method of claim 11, wherein the decrease in the number of cells of the hematopoietic system is associated with chemo- and/or radiotherapy and/or removal of blood progenitor cells.

14. The method of claim 13, wherein the chemotherapy and/or radiotherapy and/or removal of blood progenitor cells is administered to treat cancer.

15. A method for protecting hematopoeitic cells from cell death in response to a chemotherapeutic and/or radiation comprising contacting said cells with a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof prior to contacting said cells with said chemotherapeutic agent and/or radiation.

16. A method for treating a cytotoxic agent-induced hematopoietic or myeloid toxicity in a human patient which comprises administering prior to, simultaneous with or subsequent to the administration of the cytotoxic agent, a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to prevent, mitigate or reverse such hematopoietic or myeloid toxicity.

17. The method of claim 16, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in combination with one or more hematopoietic factors selected from the group consisting of Erythropoietin, G-CSF, GM- CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, stem cell factor (c-kit ligand), and M-CSF is administered in an amount effective to prevent, mitigate or reverse hematopoietic or myeloid toxicity.

18. The method of claim 17, wherein said one or more hematopoietic factors are administered prior to, concurrently with or subsequent to said administration of PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof.

19. A method of treating cancer therapy in a mammalian patient which comprises administering to the patient a combination of a cytotoxic agent in an amount effective to treat the cancer and PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to prevent, mitigate or reverse hematopoietic or myeloid toxicity, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered prior to, simultaneously with or subsequent to the administration of the cytotoxic agent, wherein administration of said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof reduces the dose of said cytotoxic agent needed to produce a therapeutic effect in said patient.

20. The method of claim 19, wherein said cytotoxic agent is a therapeutic agent.

21. A method treating cancer therapy in a mammalian patient which comprises administering to the patient a combination of a cytotoxic agent in an amount effective to treat the cancer and a combination of PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof and at least one hematopoietic growth factor in an amount effective to prevent, mitigate or reverse hematopoietic or myeloid toxicity, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered prior to the administration of the cytotoxic agent.

22. The method of claim 20, wherein said cytotoxic agent is radiation therapy, or chemotherapy.

23. The method of claim 19, wherein said mammalian patient is suffering from a cancer selected from the group consisting of Hodgkins lymphoma, Non-Hodgkins lymphoma, pancreatic cancer, melanoma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, lung cancer, Kaposi's sarcoma, leukemia, lymphoma, gastric cancer, colon cancer, colorectal cancer, esophageal cancer, renal cancer, and head and neck cancer.

24. The method of claim 21, wherein said mammalian patient is suffering from a cancer selected from the group consisting of Hodgkins lymphoma, Non-Hodgkins lymphoma, pancreatic cancer, melanoma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, lung cancer, Kaposi's sarcoma, leukemia, lymphoma, gastric cancer, colon cancer, colorectal cancer, esophageal cancer, renal cancer, and head and neck cancer.

25. The method of claim 21, wherein the hematopoietic growth factor is selected from the group consisting of Erythropoietin, G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-12, IL-13; IL-15, IL-16, IL-17, stem cell factor (c-kit ligand), and M-CSF.

26. A method for preventing or mitigating myelosuppression in a human patient undergoing therapy for cancer or tissue or organ transplantation, which comprises administering to the patient a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to treat myeloid toxicity, wherein said cytokine is administered prior to, simultaneously with or subsequent to said therapy.

27. A method for preventing or mitigating hematopoietic cell depression in a human patient undergoing therapy for cancer or tissue or organ transplantation which comprises administering to the patient a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to treat myeloid toxicity, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered prior to, simultaneously with or subsequent to the administration of said therapy.

28. A method for treating a cytotoxic agent-induced hematopoietic or myeloid toxicity in a human patient which comprises administering prior to, simultaneous with or subsequent to the administration of the therapeutic agent, PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof and a hematopoietic factor in an amount effective to prevent, mitigate or reverse such hematopoietic or myeloid toxicity.

29. The method of claim 28, wherein said cytotoxic agent is 1131, Sr89, Y90, ReI 86, Re 188, Cobalt 60; cesium 137; Iridium 192, and radium 226.

30. The method of claim 28, wherein said cytotoxic agent is conjugated to an antibody or a bone-seeking chemical.

31. The method of claim 30, wherein said bone-seeking chemical is orthophosphate or diphosphonate.

32. A method for alleviating a physical symptom caused by hematopoietic or myeloid toxicity in a human patient undergoing therapy with a cytotoxic agent, wherein the patient is administered prior to, simultaneous with or subsequent to the administration of the cytotoxic agent, a composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof in an amount effective to prevent, mitigate or reverse such hematopoietic or myeloid toxicity.

33. The method of claim 32, wherein the physical symptom is bone pain associated with bone cancer or bone metastasis.

34. The method of any of claims 1 to 33, wherein said subject is a cancer patient.

35. The method of claim 34, wherein said subject has a cancer selected from the group consisting of group consisting of Hodgkins lymphoma, Non-Hodgkins lymphoma, pancreatic cancer, melanoma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, endometrial cancer, lung cancer, Kaposi's sarcoma, leukemia, lymphoma, gastric cancer, colon cancer, colorectal cancer, esophageal cancer, renal cancer, and head and neck cancer.

36. The method of any of claims 1 to 35, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered intravenously, intraarterially, transdermal Iy, or intrathecally, or a combination thereof.

37. The method of any of claims 1 to 35, wherein the composition is formulated for administration via oral, topical, rectal, parenteral, local, intranasally, inhalant or intracerebral delivery, or a combination thereof.

38. The method of any of claims 1 to 35, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in a single dose.

39. The method of any of claims 1 to 35, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in multiple doses or as a continuous infusion during therapy.

40. The method of any of claims 1 to 35, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in a dose of about 0.5 μg/kg body weight to about lOμg /kg body weight of subject/day.

41. The method of claim 40, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered on each of 2, 3, 4, 5, 6 or 7 days prior to the administration of chemotherapy or radiation therapy.

42. The method of any of claims 1 to 35, wherein said PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is administered in combination with one or more hematopoietic factors.

43. The method of claim 42, wherein hematopoietic factor is selected from the group consisting of Erythropoietin, G-CSF, peg-G-CSF, GM-CSF, M-CSF, TCGF, flt-3, thrombopoietin, thymic stromal lymphopoietin, keratinocyte growth factor, PDGF, IGF, IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I l, IL-12, IL-13; IL-15, IL-16, IL-17, stem cell factor (c-kit ligand).

44. The method of any of claim 1 to 35, wherein said composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof comprises PTHrP 1-34, or an analog thereof.

45. The method of any of claims 1 to 35 wherein said composition comprising PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof comprises semparatide.

46. The method of claim 43 wherein the hematopoietic factor is selected from G-CSF and peg-G-CSF.

47. The method of any of the foregoing claims wherein the subject is undergoing therapy with 5-fluorouracil.

48. The method of any of the foregoing claims wherein the PTHrP, a PTHrP fragment, a PTHrP analog, or a derivative thereof is semparatide administered to the subject for a period of 4-14 days prior to the administration of myelosuppressive levels chemotherapy or radiation therapy at a daily dose of 50-200 mg/kg.