Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-09-11
2003-05-06
Romeo, David S. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S325000, C530S350000, C424S085100, C514S012200, C536S024100
Reexamination Certificate
active
06558925
ABSTRACT:
The present invention relates to variants of stem cell inhibitors.
The treatment of cancer with chemotherapeutic agents is designed to attack and destroy cells which are undergoing division within the body. A side effect of such treatment is thus the destruction of normal cells, particularly the stem cells of the haematopoietic system and the epithelial stem cells which line the scalp and gut. Radiation can also cause similar destruction of such cells.
It has been proposed that in order to improve the treatment of cancers by chemotherapy it would be desirable to protect stem cells from cell cycle specific cytotoxic drugs. WO89/10133 discloses a stem cell inhibitor and describes the use of the inhibitor in the treatment of cancers. The inhibitor may be administered to a patient in order to protect stem cells during chemotherapy.
Stem Cell Inhibitor (SCI), also known as MIP1-&agr; is a peptide of about 8 kD which forms large self aggregates, the molecular weight of which is dependant upon the concentration of SCI/MIP1-&agr; monomers (Graham et al, 1990,
Nature
344;442, Wolpe & Cerami, 1989,
FASEB J,
3; 2656). It has been found that SCI/MIP1-&agr; has a native, aggregated molecular weight of about 100 kD at 0.1 mg/ml in physiological buffers such as PBS. It has been found that diluting SCI/MIP1-&agr; to about 20-100 ng/ml or less will bring about disaggregation of this protein.
Human SCI/MIP1-&agr; has been cloned by us (Graham et al (1992),
Growth Factors
7;151-160). The cDNA has also been cloned by Nakao et al (1990,
Mol. Cell, Biol.,
10;3646-58) and called LD78&bgr;. A variant of the cDNA LD78&agr; was also found, which has a very similar sequence. It differs by only 4 amino acid residues. The human cDNA and protein sequence of the factor cloned by us is shown is Seq. ID No. 1. The first 27 amino acids are a leader sequence. The mature protein starts at residue 28 (ala). The amino acid sequence of the variant found by Nakao et al is shown as Seq. ID No. 3. The leader sequence of the protein is one amino acid shorter and thus the mature protein starts at residue 27 (ala). The sequence of the murine homologue, upon which we have conducted our work, is also known and is very similar. It can be found for example in Graham et al (1994,
J. Biol. Chem.,
269; 4974-78).
It has been reported (Mantel et al, 1993,
PNAS
90;2232) that monomeric SCI/MIP1-&agr; is more active than the aggregated form in inhibiting in vitro and in vivo stem cell proliferation. In using SCI/MIP1-&agr; in the treatment of humans it would be desirable to administer monomeric protein, not just from an activity point of view but also in order to provide reliable and reproducible formulations. However, it is likely that the low concentrations of SCI/MIP1-&agr; which must be made in order to provide monomeric protein will be too low for use in practice.
We have now surprisingly found that it is possible to obtain SCI/MIP1-&agr; variants which retain substantially the activity of the native protein but which do not form the same large aggregates. These mutants are stable as monomers or as small conglomerates (eg dimers or tetramers) at concentrations many fold higher than native SCI/MIP1-&agr;. Thus for those variants which have activity comparable to native SCI/MIP1-&agr;, the variants may have higher activity in vivo on a unit weight basis.
Accordingly, the present invention provides a Stem Cell Inhibitor protein which comprises at least one amino acid alteration from its native form which does not significantly aggregate but which retains substantially unaltered stem cell inhibitory activity. The protein may comprise either the full length stem cell inhibitor or the mature processed form lacking the leader sequence.
The invention also provides pharmaceutical compositions comprising a stem cell inhibitor according to the invention in combination with a pharmaceutically acceptable carrier or diluent, and optionally other therapeutic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof.
The formulations include those suitable for parenteral (including subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Suitable liquid carriers include phosphate buffered saline at a pH of between 7.0 and 8.0, for example 7.4. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of an active ingredient.
Formulations of the SCI/MIP-1&agr; proteins of the present invention preferably contain from 0.05 to 5 mg/ml of protein, for example 0.1 to 1.0 mg/ml. We have found that the solubility of the variants of the invention do vary although the maximum solubility of any one particular variant may be determined by simple titration by those of skill in the art.
The invention also provides such proteins and compositions for use in a method of treatment of the human or animal body.
The invention further provides a method for treating a subject who is to be exposed to an agent capable of killing dividing or cycling stem cells by administering to the subject an effective amount of a protein or composition according to the invention.
The subject may also be treated with a protein or composition according to the invention during or after chemotherapy. In the latter case, this will usually be for a period sufficient to allow clearance of the agent from the body.
The method of treatment according to the invention may be used in the treatment of solid tumours or leukemias. In the case of treatment of leukemias, it is possible to treat a sample of the patients bone marrow which has been removed from the body while the patient is undergoing treatment. The bone marrow is purged of cancer cells in the presence of a protein of composition according to the invention, and the treated marrow reintroduced into the patient.
Although the dose of the variant protein according to the invention will ultimately be at the discretion of the physician, taking into account the nature of the condition being treated and the state of the patient, effective doses may be in the range of from about 10 &mgr;g/kg body weight to about 5 mg/kg of variant protein, for example from about 50 to about 1000 &mgr;g/kg, eg about 500 &mgr;g/kg.
We have also found that SCI/MIP1-&agr; can act to enhance the expansion of primitive haemopoietic cells in ex vivo cytokine driven stem cell expansion experiments. Thus, variant proteins of the invention may also be used in methods to expand stem cell populations removed from a patient ex vivo wherein such stem cells are brought into contact with growth factors and the variant proteins of the invention under conditions which allow the growth and expansion in numbers of the cells, p
Graham Gerard
Pragnell Ian
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Romeo David S.
Wyeth
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