By Andrew J. Dart, David Hodgson and Reuben Rose,
Equine Exercise Performance Laboratory, University of Sydney, Australia

Using intra-muscular doses of PPS (Cartrophen-Vet®) up to 10 mg/kg in the horse, only two out of the 50 parameters measured showed a deviation from the expected normality range for this species for up to 168 hrs post-drug therapy. Of the two parameters (PTT and lymphocyte numbers) which did change, only PTT can be considered of possible clinical relevance since it reflects a temporary decline in the patients' haemostatic potential should it be challenged by injury or trauma. However, since no occult blood was detected in faeces or urine the transient elevation of PTT was not associated with spontaneous bleeding.

Based on the results of this study, it appears that doses of up to 3mg/kg PPS in the horse are safe and that PPS should not be administered to patients within 24 hrs of high stress activities or where physical injury may possibly be sustained, such as in show jumping. It is also evident that Warfarin or other anti-coagulant agents should not be used concurrently with pentosan polysulphate therapy.
Sodium pentosan polysulphate (PPS, Cartrophen-Vet®) is registered in Australia and elsewhere as an injectable formulation for the treatment of non-inflammatory arthropathies in the dog. It has been suggested [1] that PPS may be of potential use for the management of osteoarthritis and allied conditions in the horse, however, the safety of PPS in this species when administered intra-muscularly had not been previously described. The aim of the present study was to address this deficiency.
Eight adult horses of mixed gender, weighing between 400 and 600 kg were randomly divided into 4 groups of 2 horses each. Each group was randomly assigned to a treatment group (control = 0 mg/kg; group 3 = 3 mg/kg of sodium pentosan polysulphate (Cartrophen-Vet®); group 6 = 6 mg/kg pentosan polysulphate; group 10 = 10 mg/kg pentosan polysulphate). A Latin square design was employed to ensure that all horses received all treatments over a 4 week period as follows and as shown in Table 1.

Horses were given an anthelminthic and tetanus prophylaxis 14 days prior to beginning the experiment. Horses were brought in from the paddock the night before each treatment week and held in yards. Horses were weighed at the start of each treatment week. Horses were then brought up into the standing stocks and a 14 gauge catheter was inserted into one jugular vein, secured and flushed with saline.

Treatments were administered as a single intramuscular injection at the commencement of each week. Horses receiving 0 mg/kg of pentosan polysulphate were injected with 20 ml of sterile water. Blood was collected in a plain tube and tubes containing EDTA and sodium citrate for haematology, serum biochemistry and a clotting profile. Blood was collected in heparinised syringes for venous blood gas analysis. A faecal sample and urine sample were collected. After each sampling period horses were returned to the paddock.


Table 1

Latin square design for administration of pentosan polysulphate
(Cartrophen-Vet®) doses to horses over 4 weeks


Animals were sampled at the following times within each treatment week: 0 (pretreatment), 1, 2, 3, 4, 6, 8,24, 48 and 168 hrs post-drug administration. Data for 40 variables was collected at every time point and included:
HR: heart rate beats/min
RR: respiratory rate breaths/min
Rectal temp: rectal temperature in degrees Celsius
pH: venous blood pH
pCO2: venous partial pressure of carbon dioxide mm Hg
HCO3: venous bicarbonate mmol/L
SBE: standard base excess mmol/L
RBC: red blood cell count x 1012
Hb: haemoglobin g/L
PCV: haematocrit L/L
MCV: mean corpuscular volume fl
MCHC: mean corpuscular haemoglobin concentration g/L
MCH: mean corpuscular haemoglobin pg
WBC: white blood cell count x 109 cells/L
Neut: neutrophils x 109 cells/L
Lymph: lymphocytes x 109 cells/L
Mono: monocytes x 109 cells/L
Eosino: eosinophils x 109 cells/L
Baso: basophils x 109 cells/L
Plasma Protein: g/L
Fibrinogen: g/L
Platelets: x 109/L
PT: prothrombin time seconds
PTT: partial thromboplastin time seconds
CK: creatinine kinase U/L
AST: aspartate transferase U/L
ALP: alkaline phosphatase U/L
Tbili: total bilirubin µmol/L
Creatinine: µmol/L
Urea: mmol/L
Glucose: mmol/L
Phosphate: mmol/L
Ca: calcium mmol/L
Serum Pr: serum protein g/L
Albumin: g/L
Sodium: mmol/L
Potassium: mmol/L
Chloride: mmol/L
Lactate: mmol/L
FDP: fibrin degradation products was excluded from the data analysis since all values were 0

Data for an additional 10 variables collected at 0, 24, and 168 hr time points included:

F obl: faecal occult blood
Ursa: urinary specific gravity
Urprotein: urinary protein 1-4+
Urket: urinary ketones 1-4+
UrpH: urinary pH
UrGluc: urinary glucose 1-4+
Ur blood 1-4+
Urinary casts: number per low power field
Urinary WBC: number of white blood cells per high power field
UrRBC: number of red blood cells per high power field

All numeric values for which data was collected at every time point were analysed by a multivariate ANOVA including treatment group and time and week as fixed effects. Analyses were performed using SPSS 8.0 for Windows (alpha = 0.05). Multiple comparisons were then performed using Bonferroni tests to determine where group effects lay.

Faecal and urine samples were measured for an additional 10 variables. Five of these were numerical variables and these were analysed using a multivariate ANOVA including treatment group, time and week as fixed effects. The remaining five variables were recorded as present or absent (faecal occult blood) or on a 1 - 4 point scale. These variables were analysed using a Kruskal Wallis H test with the treatment group as a fixed effect.

The results of these analyses are summarised in Figures 1 - 41 and in Table 2, which also shows the normality ranges and 95% confidences intervals for each parameter. It was noted that the parameters heart rate, standard base excess, red blood cell count, mean corpuscular haemoglobin concentration, mean corpuscular haemoglobin, creatine kinase, urea, phosphate and sodium fell outside the normality range (see Figures 1, 7, 8, 11, 14, 25, 30, 32, 36). This discrepancy was unrelated to drug treatment since baseline values and treatment values were outside the prescribed normality range. The explanation for this discrepancy is unclear at present but some changes could be related to the response of the animals to the blood sampling procedure.

In terms of parameter change from baseline to values outside the normality range which could be attributed to drug treatment, only two parameters emerged as significant. These data are shown in Figure 16 for lymphocytes and Figure 24 for partial thromboplastin time (PTT). It is evident that lymphocyte numbers in blood of animals treated with 6 and 10 mg/kg significantly increased from baseline levels 6 hrs after drug administration but then declined to predrug values by 24 hrs (Figure 16). The estimated marginal mean values for PTT also showed a significant time-dependent increase with doses of 6 and 10 mg doses of PPS and also recovered to predrug levels by 24 hrs (Figure 24).

Following catheter placement in week 3 of the trial but prior to injection of PPS, horse 6 collapsed in the standing stocks. After approximately 1 hour the mare stood up but was blind. The mare was euthanased the following day. A full post-mortem examination was performed and the most important lesions found in the 17 year old horse were in the brain and myocardium. The brain sections revealed ischaemic damage to the temporal and occipital cerebral cortex, while the myocardium showed acute coagulative necrosis. The age of the cerebral lesions was consistent with the historical interval between the abrupt onset of tachycardia, cyanosis and blindness after placement of the jugular catheter and euthanasia the following day. The liver showed hepatocellular megalocytosis consistent with chronic hepatotoxicity referable to ingestion of pyrrolizidine alkaloids or other alkylating agents. The post mortem findings suggested that PPS administration had no bearing on the major lesions identified.

The data set from horse 6 was incomplete. Analyses were performed with and without the data set from horse 6. Inclusion of the data did not appear to effect the conclusions drawn so this data was included in the analysis.

The data collected in this study was comprehensive and included a large number of variables which were monitored for up to 168 hrs post-treatment. There were no differences detected for faecal and urinary values and these data are therefore not included. Apart from PTT and lymphocyte levels, other parameters when subjected to post hoc Bonferroni comparisons appeared to show little relationship to the treatment group effects, as evidenced by the figures which displayed the marginal means for the treatment groups as a function of time.

Partial thromboplastin time (Figure 24) did appear to exhibit a drug/dose-dependant treatment response with each increase in dose of pentosan polysulphate resulting in enhanced PTT values as measured in seconds. This finding was consistent with previous reports of the effects of pentosan polysulphate on PTT in the horse [2] and other species including man [3]. This effect which was only statistically significant when the higher doses were used was transient and returned to baseline by 24 hrs following drug administration. The elevation in lymphocyte numbers following treatment with 6 and 10 mg/kg of PPS is a well known pharmacological effect of polysulphated polysaccharides [4][5][6]. In a study with human OA patients, a transient increase in circulating lymphocytes following intramuscular injections with PPS was also reported [7]. This effect was considered to be mediated by the interaction of PPS with adhesion proteins such as the selectins which are present on cell surfaces [8]. These adhesion proteins are responsible for the processes of binding of lymphocytes to post-capillary endothelial cells and control the passage of these cells through lymph nodes. When PPS interacts with the binding proteins lymphocytes pass through lymph nodes more rapidly leading to a concomitant elevation in their numbers in the circulation for 24 hrs post-PPS therapy. This effect should be considered a useful activity since lymphocytes not only provide a source of antibodies but can also release anti-inflammatory cytokines including interleukin-4, interleukin-10 and transforming growth factor ß [9][10][11]. In this regard, it should be noted that PPS when given to animal models of inflammation at subcutaneous doses up to 50 mg/kg exhibited potent anti-inflammatory activity [12].

The blood gas analyses demonstrated a mild elevation in the pH and a lower pCO2 for several of the sampling periods in the treated animals. However, there was no consistent pattern of changes in the HCO3 with treatment. These differences could reflect a mild respiratory alkalosis in treated animals. Since there was no extreme fluctuation of values outside the normal range, these effects have little clinical relevance.

Plasma protein, serum protein and fibrinogen concentration remained within normal ranges and fluctuations did not appear to follow any pattern suggesting that pentosan polysulphate has no clinically relevant effect on these variables.

Using intra-muscular doses of PPS (Cartrophen-Vet®) up to 10 mg/kg in the horse, only two out of the 50 parameters measured showed a deviation from the expected normality range for this species for up to 168 hrs post-drug therapy. Of the two parameters (PTT and lymphocyte numbers) which did change, only PTT can be considered of possible clinical relevance since it reflects a temporary decline in the patients' haemostatic potential should it be challenged by injury or trauma. However, since no occult blood was detected in faeces or urine the transient elevation of PTT was not associated with spontaneous bleeding.

Based on the results of this study, it appears that doses of up to 3mg/kg PPS in the horse are safe and that PPS should not be administered to patients within 24 hrs of high stress activities or where physical injury may possibly be sustained, such as in show jumping. It is also evident that Warfarin or other anti-coagulant agents should not be used concurrently with pentosan polysulphate therapy.











































Table 2

The parameters which were significantly outside the normality range for each dose are shown as * (p < 0.05)

ParameterDoseResults RangeNormal Range
Studiedmg/kg(95% confidence interval)
pH 0 7.367 -
7.421
 7.32 - 7.44
3 7.373 -
7.431
6 7.371 -
7.444
10 7.366 -
7.433
pCO2 0 45.104 -
51.251
 40 - 46
3 45.009 -
51.689
6 43.874 - 51.376
10 43.624 - 52.281
HCO3 0 26.812 - 30.794 24 - 29
3 27.279 - 30.794
6 27.019 - 30.606
10 27.394 - 29.951
SBE 0 2.201 - 5.951 -1.0 - 2.0
3 2.536 - 6.326
6 2.452 - 6.173
10 2.077 - 5.298
Hb 0 126.966 - 161.564 130 - 160
3 114.281 - 163.439
6 118.428 - 149.947
10 108.928 - 155.855
PCV 0 0.314 - 0.403 0.35 - 0.44
3 0.283 - 0.408
6 0.293 - 0.369
10 0.275 -
0.385
WBC 0 5.538 -
10.611
 6.5 - 12
3 5.978 -
10.398
6 6.284 -
11.394
10 6.246 -
12.085
Lymph 0 1.639 -
3.853
 1.63 - 3.40
3 1.603 -
4.118
6 1.936 -
4.569
 *
10 2.120 -
4.661
 *
Eosino 0 0 -
0.544
 0 - 0.96
3 0 -
0.508
6 0 -
0.651
10 0.153 -
0.601
Baso 0 0 -
0.159
 0 - 0.36
3 0 -
0.176
6 0 -
0.133
10
 0 -
0.159
Plasma Protein 0 65.808 -
72.003
 60 - 73
3 65.808 -
71.274
6 65.747 -
70.503
10 65.497 -
71.503
Fibrinogen 0 2.518 -
3.539
 2 - 4
3 2.305 -
3.363
6 2.299 -
3.339
10 2.336 -
3.676
PTT 0 30.585 -
79.244
 37 - 54
3 30.210 -
103.031
6 36.006 -
162.994
 *
10 35.901 -
199.619
 *
CK 0 76.75 -
1229.498
 167 - 347
3 62.127 -
826.162
6 0 -
710.425
10 5.575 -
736.498
AST 0 218.983 -
392.635
 215 - 347
3 219.983 -
326.336
6 217.257 -
311.260
10 203.990 -
306.618
ALP 0 160.836 -
280.666
 120 - 202
3 151.336 -
237.526
6 149.209 -
223.416
10 151.834 -
283.416
Creatinine 0 95.206 -
124.529
 87 - 149
3 81.143 -
115.816
6 91.971 -
115.904
10 89.721 -
120.837
Ca 0 2.809 -
3.183
 2.78 - 3.32
3 2.730 -
3.196
6 2.933 -
3.097
10 2.962 -
3.126
Serum Protein 0 64.133 -
74.059
 60 - 70
3 64.383 -
73.100
6 63.566 -
73.809
10 66.734 -
72.934
Sodium 0 144.167 -
147.327
 134 - 142
3 142.667 -
146.523
6 142.923 -
147.952
10 141.760 -
147.077
Chloride 0 104.106 -
106.887
 97 - 105
3 103.608 -
107.441
6 103.988 -
108.512
10
103.608
-
107.137



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2. Orme CE and Harris RC: A comparison of the lipolytic and anticoagulative properties of heparin and pentosan polysulphate in the thoroughbred horse. Acta Physiol. Scand. 159: 179-185, 1997.

3. Maffrand J-P, et al. Experimental and clinical pharmacology of pentosan polysulfate. Semin. Thromb. Hemost. 17: 186-198, 1991.

4. Freitas AA and De Sousa M: Control mechanisms of lymphocyte traffic. A study of the action of two sulphated polysaccharides on the distribution of 51Cr- and [3H] adenosine-labeled mouse lymph node cells. Cell Immunol. 31: 62-76, 1977.

5. Hall JG: Sulphated polysaccharides, corticosteroids and lymphocyte recirculation. Immunology 57: 275-279, 1986.

6. Sprangrude GJ, Braaten BA and Daynes RA: Molecular mechanism of lymphocyte extravasation. I: Studies of two selective inhibitors of lymphocyte recirculation. J. Immunol. 132: 354-362, 1984.
7. Anderson JM, Edelman J and Ghosh P: Effects of pentosan polysulfate on peripheral blood leukocyte populations and mononuclear cell procoagulant activity in patients with osteoarthritis. Curr. Therap. Res. 58(2): 93-107, 1997.

8. Bevilacqua MP and Nelson RM: Endothelial-leukocyte adhesion molecules in inflammation and metastasis. Thromb. Haemost. 70: 152-154, 1993.

9. Sugiyama E, Kuroda A, Taki H, et al: Interleukin 10 cooperates with interleukin 4 to suppress inflammatory cytokine production by freshly prepared adherent rheumatology synovial cells. J. Rheumatol. 22: 2020-2026, 1995

10. van Roon JA, van Roy JL, Duits A, et al: Proinflammatory cytokine production and cartilate damage due to rheumatoid synovial T helper-1 activation is inhibited by interleukin-4. Ann. Rheum. Dis. 54: 836-840, 1995.

11. Yeh L, Augustine AJ, Lee P, et al: Interleukin-4, an inhibitor of cartilage breakdown in bovine articular cartilage. J. Rheumatol. 22: 1740-1746, 1995.

12. Ghosh P: The pathobiology of osteoarthritis and the rational for the use of pentosan polysulphate for its treatment. Semin. Arthritis Rheum. 28: 211-267, 1999.
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