Comparative study of protirelin tartrate and standard therapy in the treatment of stroke patients

cuRRENT THERAPEUTIC RESEARCH VOL.55, NO. 10, OCTOBER1994

COMPARATIVE STUDY OF PROTIRELIN TARTRATE AND STANDARD THERAPY IN THE TREATMENT OF STROKE PATIENTS GUIDO MONTANARI, PATRIZIAFRAIOLI, LIDIA MONTEMURRO, AND GIANFRANCORIZZATO Vergani Medical Division, Niguarda Ca" Granda Hospital, Milan, Italy

ABSTRACT We conducted a clinical trial to evaluate the effects of protirelin tartrate on the overtone, mobility, activities of daily living, and degree of disability of 60 patients who experienced a cerebral stroke within the 15 days preceding the start of therapy. This open, controlled study compared patients treated with protirelin plus standard therapy with those receiving standard therapy alone. The standard therapy consisted of cerebral antiedemic drugs and antiaggregants. During the first month, protirelin was administered intravenously in the hospital at a dose of 2 mg twice daily for 15 days, followed by a 15-day washout period. From month 2 through month 6, an additional five cycles of protirelin therapy given intramuscularly were administered at home. In both the protirelin and standard therapy groups, a substantial improvement was observed compared with the patients' clinical condition at baseline. This improvement was more significant in the group treated with protirelin. In particular, the difference between the protirelin and standard therapy groups for the degree of autonomy (as measured by using the Modified Parkside Behavior Rating Scale) and for mobility (as measured by using Albert's scale) reached statistical significance after 6 months of treatment. This trend was confirmed by the subjective evaluations after 3 months: most patients treated with protirelin improved, while most patients receiving standard therapy remained stable. Data on tolerability revealed no clinically significant differences between the two groups at any observation time. INTRODUCTION

Disturbances in motor activities, vigilance, and consciousness are part of the neuropsychic and neuromotor deficits that follow acute cerebral events, whether these events result from ischemic, traumatic, or hemorrhagic injuries. Patients with cerebrovascular damage exhibit remarkable alterations in neurotransmitter function, particularly in the activity of dopamine, norepinephrine, serotonin, acetylcholine, and the endorphins. 1-3 Protirelin, or thyrotropin-releasing hormone, is a neuromodulator Address correspondenceto: Dr. G. Montanari, V. Cufra 38, 20159Milan, Italy.

Received for publication on May 16, 1994. Printed in the U.S.A.

Reproductionin wholeor part is not permitted.

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PROTIRELIN TARTRATE VS STANDARDTHERAPY IN STROKE

that can enhance synaptic transmission, regulating the intensity of the neurotransmitter response. 4'5 Protirelin was first isolated in 1969 in the hypothalamus and hypophysis. It also is present in other areas of the brain and spinal cord, where it acts as a modulator on the cholinergic and monoaminergic neurotransmitter systems,6-s stimulating some specific receptors. 9,1° Protirelin tartrate is a parenteral formulation with the ability to influence learning ability and mobility. 11-1a Administration of protirelin tartrate to patients with central nervous system disturbances positively stimulates motor activities, vigilance, and consciousness. 11-15 No data are available regarding the effects of long-term administration of protirelin in humans. Thus we undertook a comparative, controlled study to evaluate the efficacy and tolerability of 6 months of treatment with protirelin tartrate in patients who had experienced a recent cerebral stroke. PATIENTS AND METHODS

From January 1990 until March 1992, we conducted an open, comparative, clinical trial to evaluate the effects of protirelin on the overtone, mobility, activities of daily living, and degree of disability of patients who had experienced a cerebral stroke within the 15 days preceding the start of therapy. A total of 60 patients between ages 55 and 75 years were enrolled in the trial. Patients with neurologic or systemic diseases that could interfere with the evaluation of the study parameters and those with clinical manifestations of dysthyroidism or hyperprolactinemia were excluded. Each patient enrolled in the study was randomly assigned to receive either protirelin tartrate plus standard therapy (n = 30) or standard therapy alone (n = 30). The Rankin scale was used to determine the severity of symptoms within each treatment group. All patients underwent a baseline computerized tomographic scan of the brain. During the first month of treatment, patients were hospitalized and received protirelin 2 mg twice daily intravenously in 250 cc of saline solution over 30 minutes for 15 days. This treatment period was followed by a 15-day washout period. During months 2 through 6, patients were treated at home with protirelin 2 mg twice daily intramuscularly. For every cycle, 15 days of treatment were followed by 15 days of washout. Standard therapy for patients with ischemic cerebral stroke consisted of antiedemic drugs (glycerol 5% 250 cc twice daily during the first 2 days and once daily during the third and fourth days) and antiaggregants (acetylsalicylic acid, indobufen, dipyridamole, or ticlopidine) and possibly furosemide to control cerebral pressure. In the presence of fluctuating disturbances (stroke in evolution), the antiaggregants therapy was replaced by calciparine 0.5 U twice daily. 1212

G. MONTANARI ET AL.

Patients with hemorrhagic strokes that were not surgically treated received 20% mannitol 2 g/kg per day or dexamethasone 8 mg twice daily. For epileptic crises, patients were treated with phenobarbital, and analgesics were given for headache. Each patient was studied for 6 months and observed at 0, 30, 60, 90, and 180 days after therapy was first administered. Informed consent was obtained from all patients. Ashworth's scale was used to measure overtone. Using this scale, we rated the various superior and inferior limb segments on a scale of 0 (no spasticity) to 4 (maximal spasticity). For the statistical analysis, spasticity was evaluated in the limb segment that, at the beginning of treatment, had the highest score. The degree of autonomy (activities of daily living) was measured by using the Modified Parkside Behavior Rating Scale (MPBRS), which yields a score ranging from 1 (normal) to 5 (complete disability) for 16 groups of behaviors observed during the daily activities of the patient. The scores were summed, with the final scores ranging from 16 (normal) to 80 (complete disability). Mobility was measured by using Albert's scale. On this scale, scores ranging from 0 (total impairment) to 5 (minimum impairment) for the 90 items that make up the following three tests: (1) test I measures mobility of the t r u n k limbs (32 items; m a x i m u m score, 170); (2) test II measures straightening, walking, and adaptation to obstacles (26 items; m a x i m u m score, 130); and (3) test III measures mobility of the upper limb (32 items; m a x i m u m score, 160). Overtone, autonomy, and mobility, as measured by Ashworth's scale, the MPBRS, and Albert's scale, were evaluated at the start of t r e a t m e n t and at the end of months 1, 2, 3, and 6 after t r e a t m e n t began. At the end of months 3 and 6, the Rankin scale (0-1 = no disability to 5 = serious disability) was used to measure the degree of patient disability, and both the patient and his or her relatives were asked to judge the efficacy of treatment. The investigator also provided his opinion of the efficacy of treatment. Vital signs were evaluated at the start of t r e a t m e n t and at the end of months 1, 2, 3, and 6. Routine laboratory tests were performed at the beginning of t r e a t m e n t and at the end of months 1, 3, and 6. Blood concentrations of T3, Tt, and thyroid-stimulating hormone were measured at the start of t r e a t m e n t and at the end of months 1, 3, and 6. At the end of therapy, the investigator gave his overall opinion of how well the drug was tolerated.

Statistical Analysis Data were gathered and reported in tables using Lotus 1-2-3 spreadsheets, then printed and double-checked. The data were then transferred to a Micro VAX II computer (Digital Equipment Co., Maynard, Massachu1213

PROTIRELIN TARTRATE VS STANDARD THERAPY IN STROKE

setts) and processed using SAS statistical software (SAS Institute Inc., Cary, North Carolina). Missing data were not included. Because the percentage of missing data was low (between 0% and 27% for measurements of clinical efficacy); the available data were sufficient to perform a statistical analysis. Hypotheses on the treatment effects were verified using nonparametric statistical tests 16 owing to the problems caused by the non-normal distribution of many data and the nonhomogeneity of variances among the various groups. Comparisons among observations made at different times ("within subjects") were made using the Wilcoxon signed-rank sum test 16 with the U N I V A R I A T E procedure of the SAS software. 17 Comparisons among observations made at the same time in the two groups ("between subjects") were made using the Wilcoxon-Mann-Whitney test 1~ with t h e N P A R l W A Y procedure of the SAS software. 17 Because of the lack of data for some patients at months 3 and 6, global comparisons (ie, Friedman's variance analysis) were not made to avoid excluding several data obtained at previous times. The critical level of statistical significance was established as P = 0.05. For multiple tests, a correction of the critical value was used according to Bonferroni's inequality, is To avoid false-positive mistakes, the critical level established was divided by the number of comparisons performed. Thus the level of significance was P = 0.017 for three comparisons, P = 0.0125 for four comparisons, and P = 0.01 for five comparisons. RESULTS

The patients in the protirelin and standard therapy groups were comparable with regard to sex, age, body weight, height, and Rankin scale score (Table I). Fifty percent of patients in both groups had a Rankin score of 2 (mild disability) and 50% had a score of 3, 4, or 5 (moderate, moderately serious, and serious disability, respectively). The number of patients studied at each time period is shown in Table II. Data from all patients enrolled in the trial were evaluable according to the study protocol. The available data were sufficient for a statistical analysis, which was performed in a w a y that reduced the possibility of making false-positive errors (type I, or alpha, statistical errors). A final Rankin scale score was available for 25 patients in the protirelin group (after 6 months for 22 patients and after 3 months for 3 patients) and for 28 patients in the standard therapy group (after 6 months for 25 patients and after 3 months for 3 patients). In both groups, the score significantly improved over time (Wilcoxon test, P < 0.001) (Table III) (Figure 1). At the end of treatment, the most significant improvement was seen in the patients in the protirelin group (Wilcoxon-Mann-Whitney test, P = 0.05). 1214

G. MONTANARI ET AL.

Table I. Patient characteristics at baseline.

Protirelin (n = 30) Sex Male Female Age (y) Median Range Body weight (kg) Mean _+ SD Height (cm) Mean _+ SD* Rankin scale score (n) 2 3,4,5

Standard Therapy (n = 30)

14 16

17 13

72 53-75

68.5 55-75

68.5 _+ 12.8

68.8 _+ 13.3

163.7 - 8.2

166.0 -+ 7.4

15 15

15 15

*Rankin scale: 0-1 = no disability, 2 = mild disability, 3 = moderate disability, 4 = moderately serious disability, and 5 = serious disability.

Spasticity receded rapidly and significantly after the first month of t r e a t m e n t (P < 0.001) and disappeared in almost all patients after 2 months (Table IV). There were no differences between groups. Initial scores on the MPBRS were comparable in the two groups and improved significantly (P < 0.0001) in both groups after the first month of t r e a t m e n t (Table V; Figure 2). The score after the first month was slightly better in the protirelin group, and this trend continued throughout the study, reaching statistical significance after 6 months (Wilcoxon-MannWhitney test, P = 0.009). The score observed at the end of treatment in the patients treated with protirelin was not significantly different from the minimum MPBRS score indicating normality. T h e scores on Albert's scale were comparable at the start of treatment and significantly improved in both groups starting from month 1 (P < 0.0001) (Table VI; Figures 3 and 4). The statistical comparison (WilcoxonMann-Whitney test) between groups after 6 months showed a value o f P = 0.041 for test I and P = 0.035 for test II. These values can be considered marginally significant, since they fall between P = 0.05 and P = 0.0125, adjusted according to Bonferroni's inequality.

Table II. Number of patients studied at each time period.

Baseline Month 1 Month 2 Month 3 Month 6

Protirelin

Standard Therapy

30 30 29 29 22

30 30 30 30 25

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PROTIRELIN TARTRATE VS STANDARD THERAPY IN STROKE

T a b l e III. F i n a l R a n k i n scale scores.*

Protirelin Final Rankin score Median Range Final Rankin score (n) 0 1 2 3 4 5

Standard Therapy

It

1.5 0-4

0-4 7 10 3 4 1 0

(28%) (40%) (12%) (16%) (4%)

1 (4%) 13 (46%) 5 (18%) 7 (25%) 2 (7%) 0

* Rankin score: 0-1 = no disability, 2 = mild disability, 3 = moderate disability, 4 = moderately serious disability, and 5 = serious diability. 1" P < 0.05 between groups.

The investigator's opinion of clinical efficacy and tolerability after 6 months was given only for protirelin (Table VII). The patients' judgment of protirelin after 6 months was favorable. Fifteen (68%) of 22 patients treated with protirelin but only 5 (20%) of 25 patients receiving standard therapy judged themselves improved or very much improved (Table VII). Only seven patients in the protirelin group, compared with 20 patients in the standard therapy group, considered their condition stable (ie, no improvement). This difference was statistically significant (Wilcoxon-MannWhitney test, P = 0.0018). The patients' evaluation was confirmed by the relatives' judgment (Table VII). According to the patients' relatives, 16 of 22 patients in the protirelin groups and 6 of 25 patients in the standard 60 EEl Protirelin E:] Standard I Therapy ]

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30

28%

25%

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18%

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12% 10

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~-~-- 4%

0

0%

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1

2

3

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4

Rankin Score F i g u r e 1. R a n k i n scale scores a t t h e e n d of t r e a t m e n t ( 0 - 1 = no disability, 2 = m i l d disability, 3 = m o d e r a t e disability, 4 = m o d e r a t e l y s e r i o u s diability, a n d 5 = s e r i o u s disability). P < 0.05 b e t w e e n groups. 1216

G. MONTANARI ET AL.

Table IV. Degree of spasticity in t h e most impaired limb.

Protirelin

Standard Therapy

Median

Range

Median

Range

2 1 0 0 0

0-3 0-3 0-3 0-3 0-1

1.5 1 0 0 0

0-3 0-3 0-2 0-2 0-1

2 1 0 0 0

0-3 0-3 0-2 0-2 0-1

2 1 0 0 0

0-3 0-3 0-2 0-2 0-1

Upper limb Baseline Month 1 Month 2 Month 3 Month 6 Lower limb Baseline Month 1 Month 2 Month 3 Month 6

therapy group were improved or very much improved. Only six patients treated with protirelin but 19 patients receiving standard therapy were judged stable by the relatives. This difference also was statistically significant (Wilcoxon-Mann-Whitney test, P = 0.0018). No significant differences were observed between the two groups at the beginning of treatment or after 1, 2, 3, and 6 months for any laboratory measurement, including thyroid function (Table VIII), or instrumental examination, with the exception of the mean systolic and diastolic blood pressure values, which were considerably decreased after the first month of treatment. Blood pressure after 6 months of treatment was significantly lower in the patients treated with protirelin compared with those receiving standard therapy (Wilcoxon-Mann-Whitney test, P = 0.0038 for systolic pressure and P = 0.004 for diastolic pressure) (Table IX). The electroencephalographic (EEG) mapping performed after 6 months remained unchanged in all patients treated with protirelin. In the standard therapy group, two EEG mappings were improved and one worsened. Table V. Scores on the Modified Parkside Behavior Rating Scale. Values are expressed as m e a n -+ SD.

Protirelin Baseline Month 1 Month 2 Month 3 Month 6

38.2 25.1 20.9 19.9 18.9

_+ 13.1 __+_9.8 _+ 7.4 _+ 7.1" _+ 6.1t

* P = 0.02 between groups. t P = 0.009 between groups. 1217

Standard Therapy 36,9 25.9 23.2 22,3 22.1

-~ 12,1 ~ 10.0 ~ 8.9 --- 7.3 -+ 7.4

PROTIRELIN TARTRATE VS STANDARD THERAPY IN STROKE

50

I

~ Protirelin ~ Standard Therapy

40

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3o

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-

-

minimum score

10 ~ 0

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1

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J 5

6

7

Figure 2. Scores on the Modified Parkside Behavior Rating Scale. *P < 0.01. DISCUSSION AND CONCLUSIONS

After 1 month of treatment, a clear improvement was seen in both groups, compared with their clinical condition at baseline. The improvement was more significant in the patients treated with protirelin. In particular, the difference between groups for the results obtained on the degree of autonomy (daily living, measured using the MPBRS) and mobility (measured using Albert's scale) reached statistical significance after 6 months of treatment. This trend was confirmed by the subjective evaluations after 3 months of treatment, in which most patients treated with protirelin judged themselves improved, while most of those receiving standard therapy judged themselves stable; these judgments were confirmed by the subjective evaluations of the patients' relatives. Table VI. Scores on Albert's scale. Values are expressed as mean -+ SD.

Test I Baseline Month 6 Percent improvement Test II Baseline Month 6 Percent improvement Test III Baseline Month 6 Percent improvement

Protirelin

Slandard Therapy

40.9 +_ 31.7 117.7 _+ 29.1 294%

43.0 + 27.3 99.6 -+ 27.5 231%

24.0 _+ 28.6 89.5 _+ 26.2 372%

27.5 _+ 30.9 71.0 _+ 25.5 258%

26.2 _+ 42.4 115.8 +_ 36.0 441%

27.2 _+ 39.5 99.9 +_ 39.6 367%

Differences between groups: P = 0.041 (test I), P = 0.035 (test II). 1218

G. MONTANARI ET AL.

160 m Protirelin I~ Standard

Therapy 120 .Q 8

80

4oi

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~

0

1

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L

L

.

2

3

4

5

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Time (mo) F i g u r e 3. Scores o n t e s t I of A l b e r t ' s scale. *P = 0.041.

No clinically significant differences in tolerability were seen between the two groups at any observation time. The difference in blood pressure values, which were significantly lower at 6 months in the group treated with protirelin (Table IX), can be explained by the more careful observation of these patients, given the concern over possible side effects, and has no clinical relevance. We observed that the patients treated with protirelin became selfsufficient, were able to care for themselves, increased their interest in their surroundings, and experienced an improvement in mood. We want to emphasize that, at the end of treatment, the MPBRS score in the group 120 m Protirelin ~ Standard1 Therapy ]

80

8

m

40

1

2

2

2

A

3 4 Time (too)

5

6

F i g u r e 4. Scores o n t e s t II of A l b e r t ' s scale. *P = 0.035. 1219

~

7

PROTIRELIN TARTRATE VS STANDARD THERAPY IN STROKE

Table VII. Subjective evaluations after 6 months of treatment.

Protirelin Clinical Efficacy Good Sufficient Tolerability Very good Good Sufficient Patient's judgment Very much improved Improved Stable Relatives' judgment Very much improved Improved Stable

Standard Therapy

21 (70%)* 1 (3%) 10 (33%) 11 (37%) 1 (3%) 1 (3%) 14 (47%) 7 (23%)

1 (3%) 4 (13%) 20 (67%)

1 (3%) 15 (50%) 6 (20%)

1(3%) 5 (17%) 19 (63%)

* The sum of percentages is lower than 100% where some data were not availablefor the analysis. Differences between groups: P = 0.0018 (patient's judgment), P = 0.0018 (relatives' judgment).

treated with protirelin (mean -+ SD, 18.9 - 6) was closer to the minimum value for normality (16 points) than the final score in the patients receiving standard therapy. We found that the degree of impairment as measured by using the M P B R S in the protirelin group decreased from 35% (38.2 --+ 13.1) to 5% (18.9 + 6.1). The improvement was less evident in the standard therapy group, in which the degree of impairment decreased from 33% (36.9 +- 12.1) to 10% (22.1 -+ 7.4). Seven (28%) patients in the protirelin group and only one (4%) patient in the standard therapy group showed a complete regression of disability, as evaluated by the Rankin scale scores. For all other values on the Rankin scale that were taken into consideration, the percentage of disabled patients was always higher in the standard therapy group. Since spasticity receded almost completely in both groups, it was not

Table VIII. Thyroid function.

Protirelin Median Thyroid-stimulating hormone Baseline End of treatment

0.9 0.9

T3Baseline End of treatment

110,5 114.0

T4Baseline End of treatment

9.4 8.9

Standard Therapy

Min

Max

0.1 0.1

2.2 1.8

63 89 7.2 6.2

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167 165 12.8 12.0

Median 0.9 0.9 118 122 8.7 9.2

Min

Max

0.3 0.4

5.4 11.3

78 77 5.6 6.0

207 206 12.7 11.8

G, MONTANARI ET AL.

Table IX. Blood pressure. Values a r e expressed as m e a n ± SD.

ProUrelin

Standard Therapy

160 ± 19.1 139 ± 8.8*

163 _+ 23.3 149 ± 11.8

88 + 8.9 81 ± 3.71"

92 + 11.9 86 +_ 4.6

Systolic (ram Hg) Baseline End of treatment Diastolicn(mm Hg) Baseli End of treatment * P = 0.038 between groups. i" P = 0.004 between groups.

possible to demonstrate any advantage for protirelin. The scores on Albert's scale indicated the superiority of protirelin for the parameters evaluated in test I (trunk/limbs) and for those evaluated in test II (straightening, walking, adaptation to obstacles); this improvement agrees with the results obtained with the MPBRS. We observed no problems with tolerability related to protirelin administration, and the decrease in the thyroid-stimulating hormone values seen in the protirelin group was neither statistically nor clinically significant, as these values always remained within normal limits. Even though the median value for thyroid-stimulating hormone was equal in the two groups (0.9), the range of values shifted toward lower values in the protirelin group (0.10 min to 1.80 max versus 0.44 min to 11.30 max in the control group). However, this imbalance was also present at baseline (0.05 rain to 2.20 max versus 0.33 min to 5.40 max in the control group). The administration of protirelin to patients suffering from stroke enabled us to obtain a more complete regression of the postictal symptoms, without any problems related to drug tolerability. The overall results show a positive action of the drug on both the neuropsychic symptoms (improvement of mood and interest in surroundings) and the various levels of motor coordination; m a n y p a t i e n t s d e m o n s t r a t e d an ability to regain selfsufficiency. Both these factors are important in the rehabilitation of the stroke patient. Patients who more quickly recover their ability to function have a better prognosis. Based on the results of our study, we recommend that protirelin be administered to patients who have experienced cerebral stroke. The use of protirelin can lead to better and faster patient recovery and decrease the social costs of assistance needed by the patient. References: 1. Moskowitz MA, W u r t m a n RJ. Acute stroke and b r a i n monoamines. In: Scheinberg P, ed. Cerebrovascular Disease. New York: Raven Press; 1976:153-165.

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PROTIRELINTARTRATEVS STANDARDTHERAPYIN STROKE

2. Pulsinelli WA. Metabolic and circulatory factors in ischemic brain injury. In: McDowell FH, Caplan LR, eds. Cerebrovascular Survey Report. 1985:75-96. 3. Siesjo BK. Cell damage in the brain: A speculative synthesis. J Cereb Blood Flow Metab. 1981;1:155-185. 4. Sharif NA. Diverse role of thyrotropin-releasing hormone in brain, pituitary and spinal function. Trends Pharmacol Sci. 1985;6:119-122. 5. Bartfai T. Presynaptic aspects of the coexistence of classical neurotransmitters and peptides. Trends Pharmacol Sci. 1985;6:331-334. 6. Emson PC, Rossor MN, Rous J, Bennett GW. The distribution and characterization of TRH in post mortem human brain. In: Griffiths EC, Bennet GW, eds. Thyrotropin Releasing Hormone. New York: Raven Press; 1983:364-369. 7. Eskay RL, Long RT, Palkovits M. Localization of immunoreactive thyrotropin releasing hormone in the lower brain stem of the rat. Brain Res. 1983;277:159-162. 8. Parker CR, Porter JC. Regional localization and subcellular compartimentation of thyrotropin releasing hormone in adult human brain. J Neurochem. 1983;41:1614-1622. 9. Manaker S, Winokur A, Rhodes CH, Rainbow TC. Autoradiographic localization of thyrotropin releasing hormone (TRH) receptors in human spinal cord. Neurology. 1985;35: 328-332. 10. Manaker S, Eichen A, Winokur A, et al. Autoradiographic localization of thyrotropin releasing hormone receptors in human brain. Neurology. 1986;36:641-646. 11. Fornarelli M, Delwaide PJ. TRH and the upper motoneuron syndrome in man. TB Today. 1988;15(Suppl 4):59-64. 12. Villardita C, Cartoni P, Cinanni G, Nicoletti G. Protirelin tartrate in the treatment of symptomatology following acute cerebrovascular insufficiency. Curr Ther Res. 1985;38: 404-413. 13. Pennisi G, Costanzo E, Cartoni P, et al. La protirelina tartrato (TRH-T) nel trattamento dell'ictus cerebri. Presse Med (Italian edition). 1988;5:13-21. 14. Interligi M, Formisano R, Fiore C, et al. Applications of TRH-T in non-degenerative diseases of the central nervous system. TB Today. 1988;15(Suppl 4):47-57. 15. Sano K, Manaka S, Hori T, et al. Clinical studies on thyrotropin releasing hormone tartrate (TRH-T) for the treatment of disturbances of consciousness (Part I). Preliminary controlled study for finding optimal doses. Jpn J Clin Exp Med. 1979;56:248-258. 16. Siegel S, Castellan NJ. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill; 1988. 17. SAS Institute Inc. SAS/STAT User's Guide. 4th ed. Cary, NC: SAS Institute Inc; 1989. 18. Glantz A. Primer of Biostatistics. New York: McGraw-Hill; 1987.

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