WO1994007524A1 - Method of treatment of arthritis with platelet factor 4 - Google Patents

Abstract

A method is provided for the treatment of inflammation, particularly inflammation of arthritis, comprising a step administering to the subject a therapeutically effective amount of human platelet factor 4, human platelet factor 4 variant 1, their pharmaceutically acceptable salts, or mixtures.

Description

  
 



   METHOD OF TREATMENT OF ARTHRITIS WITH
 PLATELET FACTOR 4
FIELD OF THE INVENTION
The present invention is directed to a method for treatment of inflammation of an arthritic condition comprising administering to a subject afflicted with such a condition a therapeutically effective amount of the protein human platelet factor 4 (PF4), or pharmaceutically acceptable salts thereof, or mixtures.



  BACKGROUND OF THE INVENTION
Human PF4, a 7800 Da protein, is present in megakaryocytes, mast cell granules and platelet alpha granules. Blood platelets, the main repository of
PF4, contain approximately 18   +    0.4   Ag    PF4/109 cells but only trace amounts (1.8   +1.0      pg/ml)    are present in human plasma. The PF4 is secreted when platelets are appropriately stimulated, e.g., when they come in contact with thrombin or damaged collagen. Mature (processed) PF4 is a 70 amino acid protein with 4 cysteine residues.



  PF4 variant 1 (PF4varl) peptide is a mature form of the human protein sequence and as used herein is intended to include all sources of the protein, including native, recombinant and chemically synthesized proteins. The protein PF4varl shows sequence diversity from PF4, particularly in a leader  sequence not found in the processed (active) forms where PF4varl has a much greater positive charge due to three additional Arg residues. The PF4varl has three amino acid differences in the mature protein sequence compared with PF4: Pro58 is Leu, Lys66 is
Glu, and Leu67 is His. The nature of these changes, all located in the carboxy-terminal region, may have a significant effect on the structure and function of
PF4varl. The carboxy-terminal region of PF4varl is, for example, less positively charged (Lys66 is Glu), which reduces its ability to bind heparin.

  The proteins PF4 and PF4varl may be made recombinantly by methods known in the art and are preferably expressed in E. coli according to methods disclosed in PCT patent application number WO 90/08824, published
August 9, 1990, incorporated by reference herein.



  It is an object of the present invention to provide a method for therapeutic treatment of inflammatory diseases or conditions by administration of PF4 and/or PF4varl.



  It is yet another object of the present invention to provide therapeutic treatment of rheumatoid arthritis.



  These and other objects will be apparent from the following description and by practice of the invention.



   SUMMARY OF THE INVENTION
The present invention provides a method for treatment of inflammation and other symptoms of arthritis comprising the step of administering to a subject a therapeutically effective amount of PF4 and/or
PF4varl, or pharmaceutically acceptable salts  thereof. The preferred method of administration is intravenous injection.



   BRIEF DESCRIPTION OF THE FIGURE
Figure 1 depicts graphical representations of the forepaw (A) and hind paw (B) thickness measurements from the experiment described in EXAMPLE 2.



   DESCRIPTION OF THE APPENDIX
SEQUENCE 1 IS THE AMINO ACID SEQUENCE OF PF4.



  SEQUENCE 2 IS THE AMINO ACID SEQUENCE OF PF4varl.



   DESCRIPTION OF THE PREFERRED EMBODIMENTS
The PF4 and PF4varl proteins, or their pharmaceutically acceptable salts, will typically be formulated with conventional pharmaceutically acceptable carriers for administration to mammals, including humans, to treat the inflammatory effects of diseases, particularly, arthritis. Suitable carriers include, but are not limited to, buffered isotonic human serum albumin solutions. The preferred mode of administration of the proteins for anti-inflammatory therapeutic purposes is by intravenous injection. The concentration of the protein that is effective for this therapeutic application varies depending upon the severity of the inflammation, the weight of the subject and the schedule of administration. The minimum effective dose of protein can be established by routine experimentation using serially diluted preparations.



  The preferred concentrations for treating humans will be within the range of about 5   pg    to 10 mg/kg per treatment. This dosage is broadly defined since the  dosage regimen may vary according to the condition to be treated, the severity thereof, and the normal variance observed from subject to subject.



  The protein may also be administered topically, for example, using conventional topical formulations such as creams, pastes, gels, sprays, ointments and salves. Carriers used in such formulations are known and include, for example, polyethylene glycol, gelatin, polyvinyl alcohol and the like. Pulmonary administration as a nasal aerosol is also useful.



  The protein may also be administered using controlled released dosage forms which typically consist of bandages or skin patches which contain the active ingredient in a manner such that it is released at a controlled rate through the skin. The control mechanism may be diffusion, osmosis, dissolution, erosion or iontophoresis. The topical formulations may contain minor amounts of additives such as emollients, stabilizers, surfactants, skin penetration altering agents and pigments. The concentration of the PF4 or PF4varl formulation in the topically administered dosage form will normally be in the range of about 0.0001% to   1    by weight of the dosage form. In any event the amount administered is sufficient to produce the desired therapeutic effect, i.e., to alleviate the inflammation caused by the arthritic condition.



  It will be realized that other variations and modifications may be made without departing from the spirit and scope of the invention. For example, an
N-terminal acylated protein may be made which may have convenient or desirable characteristics, such as, providing ease of formulation, prolonging in vivo activity, etc.  



  The following example is provided by way of illustration and is not intended to limit the invention in any way.



   Example 1
The therapeutic effect of recombinant PF4 expressed in E. coli (made as described by Barone et al.) on the progression of inflammation in established collagen-induced arthritis was tested by immunization with native type II collagen in mouse strains of the   H-29    haplotype. Histological examination of arthritic joints in collagen-induced arthritis (CIA) revealed that lesions of murine arthritis resemble those of human rheumatoid arthritis. Thus 25 male
DBA-1J mice (Jackson Laboratory, Bar Harbor, Maine) between six to eight weeks of age were immunized intradermally at the base of the tail with 100   pg    of chicken type II collagen (Genzyme, Boston,
Massachusetts) emulsified in complete Freund adjuvant (CFA) using the procedure described by Hom, et al.,
Eur. J. Immunol. 18:881-888 (1988). 

  These animals were tagged before immunization for identification.



  Control mice of 5 per group were injected with PBS.



  Animals immunized with the chicken collagen were examined three times per week for visual appearance of inflammation. Swelling was quantified by measuring the thickness of the paws, ankles, knees and wrists using a constant tension caliper. Mice were scored as arthritic if redness and swelling was observed in one paw. The first sign of swelling occurred 35 days after the primary immunization with the chicken collagen and by day 40, reached a cumulative incidence of 80% (20 out of 25). Thirtyfive days after the chicken collagen immunization, the mean hind paw thickness in arthritic mice was 3.6 + 0.1 mm, as compared to 2.5 + 0.5 mm in age-matched control mice. See Table 1.  



   Table 1
 induction of arthritis by Type   II    collagen
 Days after CII IMMUNIZATION
Mice   n=5    0 21 35 42 50 70
 Paw thickness (mm)    Arthritic   
 forepaw 1.8t0.2   2.0tO.2      23tO.2      23tO.2    2.70.3   2.8tO.3   
 hindpaw   2.3*0.3    3.00.2 3.60.1   4.OO.3    4.20.2   4.6tO.2   
 Control
 forepaw 1.80.2   2.0t0.0      2.0+0.0      2.020.0      2.020.0      2.0¯0.0   
 hindpaw   2.3s0.3      25+0.3    2.50.5   25t05    2.50.2   2.6+0.1   
DBA/U mice were immunized with CII emulsified CFA 

   on day 0 and challenged with a booster on day 21.



  Control mice were injected with PBS. Paw thickness and width were measured using a constant tension caliper. Results are expressed as meantSD.



  Subsequently, arthritic mice were allocated randomly to various treatment groups. Animals treated with rPF4 (5 per group) received i.v. injections of either 0.25 or 0.5 milligrams of rPF4/kg. body weight per day. Control arthritic mice were injected with PBS.



  Animals in each group received three weekly injections from Day 39 to the termination of the study on Day 70. The progression of the inflammatory response in each mouse was assessed by weekly measurement of paw thickness. Radiographs were taken from two animals from each group prior to immunization with chicken collagen (Day 0), on
Day 43, and at the end of the study on Day 70. After euthanasia, on Day 70, all joints distal to and including the elbow and knee, were skinned and placed in 10% buffered formalin, blindly coded and sent for histological evaluation. Histological changes were graded based on the degree of cartilage erosion, edema, vascularity, synovial lining, cell  hyperplasia, papillary synovia, pannus, as well as plasma cell polymorphonuclear leukocyte lymphocyte and macrophage infiltrations. Each extremity was graded on a scale of zero to three.



  The inflammation in PBS-treated arthritic mice progressed considerably with time. The mean hind paw thickness in this group increased from 3.6   +    0.1 mm to 4.6 + 0.2 mm from Day 35 to Day 70 (see Table 1).



  Four weeks of treatment with 0.5 milligrams of
PF4/kilogram body weight reduced the paw swelling back to normal. At the end of the study, the mean hind paw thickness of animals treated with 0.5 mg of
PF4/kg. body weight was 2.7   +    0.2 mm (Table 2) as compared with 2.6   +    0.1 mm in the age-matched normal mice (Table 1).



   TABLE 2
 Effect of rPF4 treatment on paw swelling in arthritic mice
Weeks rPF4 after rPF4 ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ therapy PBS 050 mg/kg 0.25 mg/kg
 Paw thickness (mm)
 2 forepaw   2.3+0.2      2.0+0.2      2.320.1   
 hindpaw   4.0tO.3      3.3t0.3c      3sot0.2   
 3b forepaw   2.7tO.3      1.8+0.3      2.0t0.1   
 hindpaw   4.2+0.2      2.9t0.id      3.3+0.2C   
 4b forepaw   2.8tO.2      1.8+0.3      2.0+0.2   
 hindpaw 4.6t0.2   2.7t0.2d      3.1t0.id   
Thirty nine days after the primary immunization with  <RTI  

    ID=7.21> Cull,    five mice per group received three weekly injections of rPF4. Control mice were injected with PBS. Paw thickness and width were measured using a constant tension caliper. Results are expressed as meantSD.



  bn=3   CP < 0.05;      dP < 0.001;      Cp < 0.005    by two-tailed t test.



  Radiologic evaluation on Day 70 reveals distinct periosteal and degenerative arthritic changes in the
PBS-treated arthritic mice, however mice treated with three weekly doses of 0.5 mg of PF4/kg per day had  minimal signs of bony reaction. Histological examination of the limbs of arthritic mice reveals variation in the intensity of hyperplastic and inflammatory synovial lesions between the right and left limbs and between the joints of any given limb.



  Lesions were most severe in the hind limbs of PBStreated arthritic mice as compared to the hind limbs from animals treated with 0.5 mg of the rPF4/kg body weight. The lesions in animals treated with 0.25 mg/PF4/body weight were identifiable but milder as compared to the PBS-treated arthritic mice. The PBStreated arthritic mice exhibited marked to moderate hind limb hyperplastic non-suppurative synovitis characterized histologically by the thickening and/or redundant folding of synovium with variable infiltrates of mononuclear cells which were predominately macrophages and lymphocytes. The lymphocytic inflammatory infiltrates were confined to the synovium not extending into periarticular soft tissues. There was moderate thickening of the synovia, particularly those with marked leukocytic infiltrates. However, there was no marked alteration in bone density.



   ExamPle 2
Another experiment was carried out in which groups of five arthritic mice were treated with three weekly doses of either rPF4 or PF4 variant 1 (PF4 varl).



  PF4 varl, a naturally occurring variant of human PF4, is identical to PF4 except for three amino acid differences in the carboxy-terminal domain (Green et al., Mol. Cel. Biol.   9:1445-1451    (1981)). In parallel experiments, the effects of   ss-thromboglobulin      (BTG),    another heparin-binding protein that shares 50% homology with PF4, (Begg et al., Biochem. 17:1739-44 (1987)), was evaluated on  the progression of CIA. Animals in the control group were injected with PBS.



  To induce arthritis, male DBA/IJ mice were immunized with chicken type II collagen (CII) using the previously described procedures. After the visual appearance of arthritis (erythema and swelling in at least one paw) groups of five randomly assigned arthritic mice were injected intravenously (i.v) for three consecutive days per week with either 0.5 mg/kg body weight per day recombinant PF4 (rPF4) or 0.5 mg/kg per day PF4 variantl (PF4 varl) dissolved in 0.5 M phosphate buffered saline (PBS), pH 7.2.



  Control arthritic mice received either 0.5 mg/kg per day   8-thromboglobulin    (BTG), or PBS. Treatment started on day 42, after the visual appearance of the disease, and was continued for four weeks. Swelling was quantified by measuring paw thickness, as well as the width of elbow, ankles, and knees with a constant tension caliper. The data were recorded as the mean + SD of the measurements for any given limb.

 

  Four days after the final injection, mice were euthanized and body weight was recorded. Examination of various organs revealed no abnormalities except for a slight enlargement of the deep inguinal lymph nodes. Both the front and hind limbs from each animal regardless of their clinical involvement were excised and joints distal to and including the elbow or the knee were skinned and fixed in 100% neutral buffered formalin. Each limb was decalcified and sectioned longitudinally. Each longitudinal half was embedded in paraffin, sectioned, and stained with hematoxylin and eosin. To ensure examination of representative area, multiple level(s) sections from both the forepaws and the hindpaws including the interphalangeal joints, carpal/tarsal joints, and  metacarpal/metatarsal joints were evaluated.

  Each limb was graded, in comparison to that from the age-matched non-arthritic mice, on a scale of   0    to 3 for the severity of arthritis on the basis of the following changes: 1) cartilage erosion; 2) edema; 3) plasma cells; 4) polymorphonuclear leukocytes; 5) lymphocytes; 6) macrophages; 7) vascularity; 8) fibrin deposition; 9) gaint cells; 10) synovial cell proliferation; 11) lymphoid follicles; 12) papillary synovia; 13) pannus; 14) joint distortion; 15) bony destruction, remodeling, periosteal proliferation; and 16) periarticular fibroplasia. Arthritic index for each mouse was calculated by the following equation:
 Sum of the scores for the four limbs
 No. of parameters evaluated
Arthritic index of 0 represents no detectable lesion, and 12 the highest possible score.



  The first visual appearance of arthritis (erythema and paw swelling) was observed between 28 to 35 days after the primary immunization with CII. Forty-two days after immunization with chicken type II collagen, the mean forepaw and hindpaw thickness of the arthritic mice was 2.6   +    0.5 mm and 3.7   +    0.2 mm, respectively. Paw swelling in the PBS-injected arthritic mice increased with time. At the completion of the study (day 70), the mean forepaw and hindpaw thickness in this group was 3.1   +    0.2 mm and 4.1 + 0.2 mm, respectively (Figure 1). The data are presented as the mean + SD. Differences between the groups were analyzed by ANOVA, followed by the
Newman-Keul's test.



  Systemic administration of PF4-based compounds significantly suppressed paw swelling (P < 0.0 1)   (Figure 1) and effectively reduced the severity of arthritic lesions reflected by the arthritic indices compared to the PBS-injected arthritic mice (Table 3). By histological parameters, treatment with the 0.5 mg dosage of BTG had no effect on the progression of the disease (arthritic index of 3.1   +    7 versus 3.2   +    0.5 in the PBS-injected arthritic group).



  Arthritic lesions in mice injected with PBS were often present unilaterally but in multiple joints and were quite advanced in their pathologic features.



  Synovium of these animals displayed a consistent infiltration of mononuclear inflammatory cells and predominantly macrophages and lymphocytes, an exuberant periosteal proliferation, accompanied by periarticular fibrosis, distortion of the joint and disruption of joint integrity. The inflammatory cell infiltrates were confined to the synovium, joint space and bony structures, and did not extend into periarticular soft tissues. Limbs from animals treated with rPF4 showed no or minimal inflammatory cell infilteration and cartilage erosion compared to
PBS-injected arthritic mice (Arthritic index of   0    vs 3.2 + 0.5).

  Treatment with PF4 varl also suppressed paw swelling (P < 0.01) and greatly reduced synovial inflammation in three of the four mice examined histologically (arthritic index ranging from 0 to 0.8 versus 3.2   +    0.5 in the PBS-injected arthritic mice).



  The arthritic index in the nonresponder mouse in animals treated with PF4   Varl    was 2.6.  



   TABLE 3
Therapeutic Effect of PF4-based Compounds on
Collagen-Induced Arthritis
Treatment Arthritic Indexa
PBS 3.2 + 0.5 rPF4 (0.5 mg/kg) 0
PF4 - var 1(0.5 mg/kg) 0.85 +   12b   
BTG 3.1 + 0.7 a Both the forepaws and the hindpaws including the
 interpha- langeal joints, the carpal/tarsal, and
 metacarpal/metatarsal joint spaces were examined.



   Each limb was graded on a scale of   0    to 3 for the
 severity of arthritis.



  Arthritic index of 0 represents no detectable disease.



  b One of the four animals tested had an arthritic
 index of 2.6.



  Having disclosed the preferred embodiments of the invention, it will be apparent to those of ordinary skill in the art that various modifications and variations may be made without departing from the spirit and scope of the invention. The invention is not deemed to be limited in any way by the foregoing description of the preferred embodiments.  



   SEQUENCE LISTING (1) GENERAL INFORMATION:
 (i) APPLICANT:
 Nahid Mohagheghpour
 (ii) TITLE OF INVENTION:
 METHOD OF TREATMENT OF ARTHRITIS
 WITH PLATELET FACTOR 4
 (iii) NUMBER OF SEQUENCES: 2
 (iv) CORRESPONDENCE ADDRESS:
 (A) ADDRESSEE:
 Heller, Ehrman, White  & McAuliffe
 (B) STREET:
 333 Bush Street
 (C) CITY:
 San Francisco
 (D) STATE:
 California
 (E) COUNTRY:
 United States of America
 (F) ZIP:
 94104-2878
 (v) COMPUTER READABLE FORM:
 (A) MEDIUM TYPE:
 Diskette, 3.50 inch, 1.2 Kb storage
 (B) COMPUTER:
 IBM PC/XT/AT compatible
 (C) OPERATING SYSTEM:
 MS Dos 3.3
 (D) SOFTWARE:
 Word Perfect 5.1
 (vi) CURRENT APPLICATION DATA:
 (A) APPLICATION NUMBER:
 (B) FILING DATE:  
 (C) CLASSIFICATION:
 (viii) ATTORNEY/AGENT INFORMATION:
 (A) NAME:
 Reginald J.

  Suyat
 (B) REGISTRATION NUMBER:
 28,172
   (C)    REFERENCE/DOCKET NUMBER:
 11561-0016
 (ix) TELECOMMUNICATIONS INFORMATION:
 (A) TELEPHONE:
 (415) 772-6432
 (B) TELEFAX:
 (415) 772-6268
 (C) TELEX:
 184-996 (2) INFORMATION FOR SEQ ID NO:1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH:
 70 amino acids
 (B) TYPE:
 amino acid
 (D) TOPOLOGY:
 Linear
 (ii) MOLECULE TYPE:
 (A) DESCRIPTION:
 -protein
 (ix) FEATURE:
 (A) NAME/KEY: hPF4
 (x) PUBLICATION INFORMATION:
 (A) AUTHORS: Ponz, M. et al.



   (B) TITLE:
 (C) JOURNAL: Blood
 (D) VOLUME: 69
 (E) PAGES: 219 ff
 (G) DATE: 1987  
 (xi) A SEQUENCE DESCRIPTION:SEQ ID NO:1:
Glu Ala Glu Glu Asp Gly Asp Leu   Gln      cys    Leu   cys    Val Lys Thr
 5 10 15
Thr Ser   Gln    Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile
 20 25 30
Lys Ala Gly Pro His Cys Pro Thr Ala Gln Leu Ile Ala Thr Leu
 35 40 45
Lys Asn Gly Arg Lys Ile Cys Leu Asp Leu Gln Ala Pro Leu Tyr
 50 55 60
Lys Lys Ile Ile Lys Lys Leu Leu Glu Ser
 65 70   (2) INFORMATION FOR SEQ ID NO:2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH:
 70 amino acids
 (B) TYPE:
 amino acid
 (D) TOPOLOGY:
 Linear
 (ii) MOLECULE TYPE:
 (A) DESCRIPTION:
 -protein
 (ix) FEATURE:
 (A) NAME/KEY: PF4varl
 (x) PUBLICATION INFORMATION:
 (A) AUTHORS: Chen, J.Y.

  

   (B) TITLE: Cloning and Expression of
 a Variant Gene of
 Platelet Factor 4 and
 Compositions Thereof to
 Modulate Immune Responses
 (H) DOCUMENT NUMBER: PCT/US90/00320
 (I) FILING DATE: 18 JAN 1990
 (J) PUBLICATION DATE: 9 AUG 1990  
 (xi) A SEQUENCE DESCRIPTION:SEQ ID NO:2:
Glu Ala Glu Glu Asp Glu Asp Leu   Gln    Cys Leu Cys Val Lys Thr
 5 10 15
Thr Ser   Gln    Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile
 20 25 30
Lys Ala Gly Pro His Cys Pro Thr Ala Gln Leu Ile Ala Thr Leu
 35 40 45
Lys   Asn    Gly Arg Lys Ile Cys Leu Asp Leu Gln Ala Leu Leu Tyr
 50 55 60
Lys Lys Ile Ile Lys Glu His Leu Glu Ser
 65 70 

Claims

WHAT IS CLAIMED IS: 1. A method for treatment of inflammation in a subject comprising the step of administering to said subject a therapeutically effective amount of a protein selected from the group consisting of human platelet factor 4, human platelet factor 4 variant 1, their pharmaceutically acceptable salts, and mixtures of said factors and/or said salts.

2. A method according to Claim 1 wherein said inflammation is a symptom of an arthritic condition in said subject.

3. A method according to Claim 1 or 2 wherein said protein comprises human platelet factor 4.

4. A method according to Claim 1 or 2 wherein said protein comprises human platelet factor 4 variant 1.

5. A method according to Claim 3 wherein said protein is a recombinant form.

6. A method according to Claim 4 wherein said protein is a recombinant form.

7. A method according to Claim 2 wherein said arthritic condition comprises rheumatoid arthritis.

8. A pharmaceutical composition for treatment of inflammation in a subject, comprising a pharmaceutically acceptable carrier and an inflammation-alleviating effective amount of a protein selected from the group consisting of human platelet factor 4, human platelet factor 4 variant 1, their pharmaceutically acceptable salts, and mixtures of said factors and/or said salts.

9. A composition according to Claim 8 which said inflammation is a symptom of an arthritic condition in said subject.

10. A composition according to Claim 8 or 9 wherein said protein comprises human platelet factor 4.

11. A composition according to Claim 8 or 9 wherein said protein comprises human platelet variant 1.

12. A composition according to Claim 10 wherein said protein is a recombinant form.

13. A composition according to Claim 11 wherein said protein is a recombinant form.

14. A composition according to Claim 9 wherein arthritic condition comprises rheumatoid arthritis.