~

Cancer ImmunolImmunother(1991) 34: 169-174

ancer mmunology mmunotherapy

© Springer-Verlag 1991

Pharmacokinetics and biological activity in subcutaneous long-term administration of recombinant interferon-y in cancer patients W. Digel 1, G. Zahn2, G. Heinzel 2, and F. Porzsolt 1 1Departmentof InternalMedicineIII, Universityof Ulm, 7900 Ulm, and 2 Dr. Karl ThomaeGmbH,7950 Biberach,FederalRepublic of Germany Received 16 May 1991/Accepted3 September 1991

Summary. We have investigated the pharmacokinetics, tolerance, and biological activity of recombinant human interferon-7 (rHuIFN7) administered subcutaneously to cancer patients. Twenty-one patients with lymphoma and metastatic cancer received rHuIFN 7 (in doses of 0.1, 0.25, or 0.5 mg/m 2) in two or three injections per week for up to 180 days. The most common adverse effects encountered were flu-like symptoms, fever and fatigue. The increase in body temperature after each administration ranged from 0 to 4°C depending on the individual patient, but was unrelated to the rHuIFN 7 dose or its plasma concentration. The pharmacokinetic response of the patients after the two treatments showed a low intra-individual variability with respect to the plasma concentration/time profiles. However, as observed for the fever side-effect, the interindividual variation (CV >50%) was high for the parameters area under the data points (AUC0_ t) and maximum plasma concentration (Cmax). Despite this high interindividual variability, the mean values obtained for AUC0-t and Cmax after s. c. injection of rHuIFN7 were approximately proportional to the dose administered: the injection of 0.1, 0.25 or 0.5 mg/m2 rHuIFN~{ resulted in AUC0-t values of 15.4, 31.5 or 69.6 ng h/tal, respectively and Cmax was found to be 1.0, 2.4 and 4.9 ng/ml, respectively. With this s. c. administration protocol, objective antitumour responses were observed in two patients, but there was no partial or complete remission. Key words: Recombinant interferon- 7 - Pharmacokinetics - Metastases

Offprint requests to: W. Digel, Departmentof InternalMedicine I (Haematology/Oncology),Albert-Ludwigs-Universityof Freiburg, Hugstetter-Str. 55, D-7800 Freiburg,FRG

Introduction Interferons are naturally occurring inducible proteins that are produced by eucariotic cells in response to different stimuli with potent antiviral, antiproliferative and immunomodulatory effects [2]. There are two major families of human interferon; these include the large family (type I) of leucocyte interferons (IFNo0 and fibroblast interferon (IFN~), all of which are structurally related, virally induced, acid-stable molecules, which use the same receptor [28]. In contrast, type II IFN is an acid-labile glycoprotein produced by activated T lymphocytes in response to antigen, mitogen or other cytokines [14]. Antitumour activity in vivo in several cancers has been demonstrated in clinical investigations of partially pure IFN(z and IFN~, as well as recombinant IFNo~ [7, 8, 17, 20, 25, 26, 29]. In vitro studies have shown that IFNy shares many functional properties with the other interferons, but there are important differences in their biological spectra of action, e. g. immunoregulatory activities [1,27]. Therefore, IFNy may possibly have significant clinical implications as an antitumour agent. IFNy induction of macrophage activity possesses both tumoricidal and microbicidal properties [21]. IFNy also has the ability to differentiate both normal and tumour cells [23, 24], and it can induce specific receptors for many cytokines [31] or antigens [11]. Furthermore it has been shown that IFNy is a potent inducer of cytotoxins [22]. The implications of such effects for the in vivo situation is not well understood, although, a great therapeutic potential is ascribed to IFNy as a "biological response modifier". Because of the highly species-specific nature of that lymphokine, safety and efficacy evaluation has to rely solely on human studies. IFNy recently became available for clinical trials in the form of recombinant human interferony (rHuIFNy), which is a non-glycosylated protein (17 500 Da). In this study, the tolerability and pharmacokinetics of rHuIFNT after subcutaneous (s. c.) administration were investigated, and there was a first evaluation of its efficacy.

170

Table 1. Patient characteristics Sex

Male Female

19 2

Age (years) Median Range

57 16-77

Diagnoses Renal cell carcinoma Lymphoma Colon carcinoma Liver cell carcinoma Neurosarcoma Osteosarcoma Pancreatic carcinoma

10 5 2 1 1 1 1

Materials and methods rHuIFNy produced in E. coli [6] and prepared as a sterile lyophilized powder (IF-RC 1001 XX G, Thomae, Biberach, FRG) was reconstituted with sterile water immediately before injection to a final concentration of 0.5 mg/tal or 1.0 mg/ml. The specific activity was approximately 2 x 107 IU/mg protein. Patient selection and study design. The patients selected for this study all had histologically diagnosed malignancies resistant to conventional therapy (lymphomas, solid tumours). The main inclusion criteria comprised a Karnofsky performance status of more than 50%, no past anticancer therapy within 4 weeks prior to this investigation and a life expectancy of more than 3 months. Inlbrmed consent was obtained in accordance with the institutional policy. The study was designed as an open, interindividual comparison of three rHuIFNy dose levels administered subcutaneously. Each of the three dosage groups consisted of 6 - 7 patients. The first administration (tolerability, phannacokinetics) was followed by a second one after 1 week (on days 5 - 9 , pharmacokinetics) and subsequently by two to three treatments per week for up to 26 weeks (long-term tolerability, efficacy). For the first 14 days the patients were treated in the hospital, thereafter they were monitored as out-patients. Tolerance and efficacy assessment. A complete blood count, electrolytes and a clinical chemistry profile were obtained prior to the study, twice a week during hospitalization, and then every 2 weeks thereafter. An etectrocardiogram and a chest X-ray were performed before the study, every 4 weeks during the study, and afterwards. Local tolerability of the medication was assessed by visual inspection of the injection sites after each treatment. Any other symptoms were recorded as mild, moderate, or severe. Mild referred to symptoms that cansed no change in the performance levels and did not require medication for relief. Moderate symptoms were those that required medication for relief. Severe signified symptoms that were inadequately relivered by medication and caused a greater than 25% drop in performance status. Changes in body temperature were also classified according to mild (37-38 °C), moderate (38 39 °C), and severe (>39 ° C). A bioassay for the detection of neutralizing antibodies against IFNy was performed as previously described [4]. All patients were tested before therapy, 2 weeks after initiation, and 4 weeks after cessation of therapy. Tumour size was evaluated in haematological malignancies by a peripheral blood cell count or haematological improvement. In patients with solid tumours tumour size was evaluated by radiological studies and scans. Clinical pharmacology. Venous blood samples were collected from an antecubital vein into tubes containing NH4-heparin (7.5 I. E. heparin/ml blood, Saarstedt Nürnbrecht, FRG). After centrifugation of the blood

samples, the plasma was decanted and stored at -20 ° C until analysis. The first set of blood samples were collected prior to injection of rHuIFNy, a second set 2, 4, 8, 10, 12, 24, 32, 48, and 72 h after the first s. c. dose, and the rinal set 4, 8, 12, 24, 32, and 72 h after the second injection, rHuIFNy plasma levels were monitored by using a modified Centocor radioimmunoassay (Centocor, Malvern, Pa., USA). The modifications were as follows: Serodos Ingotest 58 09 51 (Boehringer, Ingelheim, FRG) was used for the dilution of the standard and of samples instead of the "0-standard" provided by Centocor. In addition, a rHuIFNy standard was used that had been adjusted to an IFNyreference standard Gg 23-901-530 (N1H, Bethesda, Md., USA). The following pharmacokinetic parameters were determined: AUC0-t (area under the data points), Cmax(maximum plasma concentration), tma× (time to reach maximum plasma concentration), and MRT (mean residence time). The mean residence time represents the average time spent by a molecule of a drug or metabolite in the "compartment" under examination (plasma, whole body). For example, after s. c. administration, the overall MRT for a compound (total MRT or MRTtot) can be represented as the sum of the MRT for absorption (MRTabs) and the MRT for disposition (intrinsic MRT or MRTint). AUC0 t for each of the two administration intervals was calculated between the first and the last data points according to the trapezoidal rule. The Cma×values and the AUC0 t values of the 0.1 mg/m2 and 0.25 mg/m2 dosage groups were normalized to the highest dose of 0.5 mg/rn 2 in order to demonstrate dose proportionality and for statistical calculations, tmax and total MRT were calculated using the Topfit nonlinear regression program [10]. An open one-compartment disposition model was applied for pharmacokinetic analysis. The plasma concentration/time curves after the first and second treatments were fitted separately as well as together in one run for each patient. For the analysis of variance and for the multiple-group comparison, standard program packages were used [5].

Results T w e n t y - o n e p a t i e n t s w e r e e n r o l l e d in this study in w h i c h a total o f 422 r H u I F N y i n j e c t i o n s w e r e a d m i n i s t e r e d . Patient c h a r a c t e r i s t i c s are d e p i c t e d in T a b l e 1. T h e m e a n d u r a t i o n o f a d m i n i s t r a t i o n was 92 days ( r a n g e 3 - 1 8 0 d a y s ) . P a t i e n t s w i t h renal cell c a r c i n o m a r e p r e s e n t e d a b o u t 5 0 % o f the p a t i e n t s treated.

Tolerability W e d i d n o t n o t i c e any l o c a l r e a c t i o n at the site o f the i n j e c t i o n . L i v e r and r e n a l f u n c t i o n tests r e m a i n e d unc h a n g e d t h r o u g h o u t the study e x c e p t f o r a m o d e r a t e inc r e a s e in s e r u m aspartate a m i n o t r a n s f e r a s e in 1 p a t i e n t and an a l k a l i n e p h o s p h a t a s e i n c r e a s e o f up to t w o t i m e s baseline v a l u e in 3 patients. C h o l i n e e s t e r a s e d e c r e a s e d in 1 p a t i e n t to a p a t h o l o g i c a l value. T h e clinical s i d e - e f f e c t s a s s o c i a t e d w i t h r H u I F N y are s h o w n in T a b l e 2. O n e patient with pancreatic carcinoma died of thromboembolic comp l i c a t i o n s 3 d a y s after the first treatment. T h e m a j o r i t y o f i n j e c t i o n s r e s u l t e d in fever. F e v e r b e g a n 1 h after s. c. injections a n d p e a k e d at 6 - 1 2 h. T h e r e w a s n o c o r r e l a t i o n bet w e e n the e x t e n s i o n o f f e v e r and the a d m i n i s t e r e d r H u I F N y d o s e (Fig. 1). D u r i n g the l o n g - t e r m a d m i n i s t r a t i o n the deg r e e o f f e v e r in all e x a m i n e d c a s e s w a s c o n s t a n t (Fig. 1). O n l y m i n i m a l c h a n g e s in p e r i p h e r a l b l o o d w h i t e b l o o d cell count, platelets, and H g b w e r e s e e n d u r i n g the c o u r s e o f t h e r a p y . A c c o r d i n g to the c l i n i c a l p r o t o c o l , the l e v e l o f

171 Table 2. Nonhaematological toxicity after s. c. administration of recombinant human interferon 7 (rHuIFNy) to 7 patients at each dose Toxicitya

No. patients showing symptoms for a dose of rHuIFNy of

['el

..

~

P20/0.1 mg

.°1

0.1 mg/m2

0.25 mg/m2

0.5 mg/m2

Fever Mild (37 -38 °C) Moderate (38- 39°C) Severe (>39°C)

2 2 2

0 2 1

1 3 0

Chills Mild Moderate Severe

3 0 0

1 1 0

2 0 0

Headache Mild Moderate Severe

2 0 0

4 0 0

2 1 0

Fatique Mild Moderate Severe

0 0 0

1 1 0

1 0 0

Fig. 1. Extent of fever after s.c. administration of recombinant human interferon 7 (rHuIFNT). The curves represent the maximum temperatures reached by different patients and after different dosages during l ong-term administration of rHuIFN7. 0 - - 0 , patient 20 (0.1 mg/m2); • •, patient 10 (0.5 mg/m2); • • , patient 15 (0.25 mg/m2); O O, patient 7 (0.5 mg/m2)

Nausea and vomiting Mild Moderate Severe

1 0 1

0 1 0

0 0 0

Pharmacokinetics

Sleeplessness Mild Moderate Severe

0 1 0

0 0 0

0 0 1

Diarrhoea Mild Moderate Severe

0 1 0

0 0 0

0 0 0

a The symptoms were recorded as mild, moderate or severe as described in Materials and methods (Tolerance and efficacy assessment)

antibodies neutralizing the antiviral activity o f r H u I F N 7 was determined in plasma samples prior to administration as well as 2 weeks after beginning therapy and 4 weeks after cessation. None of the 21 patients developed neutralizing antibodies to r H u I F N y (data not presented).

Antitumour activity Neither a partial nor a complete response was seen in any of the patients treated with r H u I F N 7 in this study. One patient with renal cell carcinoma, treated with 0.25 m g / m 2, demonstrated a measurable mixed response in his lymph nodes. Another patient with osteosarcoma, treated with 0.25 m g / m 2, had a marked regression o f a lung metastasis, whereas a new lung metastasis developed during further treatment. The disease stabilized in 2 more patients, but in all other patients the disease progressed.

Admlnisntions

The pharmacokinetic parameters A U C 0 - t , Cmax, tmax and M R T were calculated from the plasma level/time curves after the first administration o f r H u I F N ? and after the second administration. The areas as well as the m a x i m u m plasma concentrations obtained after injecting 0.1 m g / m 2 and 0.25 m g / m 2 r H u I F N 7 were normalized to the highest dose administered (0.5 m g / m 2) in order to determine dose proportionality. Table 3 summarizes the pharmacokinetic parameters and demonstrates that the data obtained after the first administration are comparable to those after the second administration. Below, we will describe the data found after the first administration only: the mean normalized A U C 0 - t values ( x + S D ) for the doses o f 0.1 mg/m2 (n = 6) and 0.25 m g / m 2 (n = 7) were 7 7 . 2 + 5 9 . 9 ng h/ml (range: 3 2 . 8 - 1 9 3 ng h/ml) and 62.9 + 17.4 ng h/ml (range: 3 1 . 3 - 8 0 . 3 ng h/ml) respectively. The administration of 0.5 m g / m 2 (n = 6) resulted in an AUC0 _ t value (x___SD) of 6 9 . 2 + 2 7 . 6 ng h/ml (range: 3 9 . 9 - 1 1 7 . 4 ng h/ml). There was no significant difference between the normalized mean areas o f the three dosage groups when a multiple group comparison was performed. The mean normalized Cmaxvalues (x + SD) for the doses of 0.1 mg/m2 (n = 6) and 0.25 mg/m2 (n=7) were 4.6 + 3.8 ng/ml (range: 0 . 8 - 1 1 . 7 ng/ml) and 4 . 8 + 1.6 ng/ml (range: 2 . 3 - 7 . 1 ng/ml) respectively. The administration of 0.5 m g / m 2 (n = 6) resulted in mean Cmax values (x + SD) of 4.9 + 2.9 ng/ml (fange: 2.7 - 9.8 ng/ml). Again, there was no significant difference between the three dosage groups with respect to the normalized mean Cmax values when a multiple group comparison was performed. The pharmacokinetics was shown to be dose-proportional with respect to the parameters A U C 0 - t and Cmax. For the parameters tmax and total M R T the means were calcu-

172 Table 3. Individual pharmacokinetic parameters after the first (1 st) and after the second (2nd) subcutaneous administration of 0.1, 0.25, and 0.5 mg/m2 rHulFN 7 to patientsa Dose

0.10 mg/m2

Patient no.

AUC (ng h/ml) norm. to 0.5 mg/m 2

tmax (h)

1st

1st

2nd

01 02 08 11 19 20 21

39.1 45.6 69.8 193.0 32.8

217.7 23.8

82.9

0.25 mg/m2

03 04 05 09 15 17 18

0.50 mg/m2

07 10 12 13 14 16

n Mean SD CV (%)

36.9 34.8

91.3

4.29 6.58 2.95 8.24 5.89 7.87 8.06

70.0 51.0 80.3 73.7 57.8 76.5 31.3

54.2 37.4 70.5 75.5 60.5 61.0 14.9

55.0 66.3 82.5 54.1 39.9 117.4

76.5 101.5 87.6 67.2 51.0 107.5

19 69.42 36.66 52.81

18 70.54 44.99 63.77

2nd 8.00 8.09

Cmax(mg/m2) norm. to 0.5 mg/m 2

MRT (h)

1st

1st

2nd 12.12 10.32

2nd

9.00 9.11 8.00 7.84

2.40 2.75 7.35 11.65 2.25 0.75 4.85

2.50 2.45 11.60 0.95 0.85 5.30

13.28 12.64 7.66 10.26 8.31 12.25 12.50

6.33 3.65 4.42 8.75 3.02 6.11 4.93

8.81 8.00 8.00 9.00 4.42 5.95 5.01

4.18 6.32 7.06 4.20 4.40 5.32 2.32

3.86 4.00 5.52 4.86 3.48 4.42 1.12

12.33 6.14 9.74 13.15 6.52 9.29 10.79

10.31 7.68 10.37 10.66 8.46 8.80 8.95

6.84 9.76 8.11 8.75 6.77 6.07

8.00 7.35 6.58 10.35 10.84 5.95

3.32 3.83 7.13 2.68 2.73 9.79

5.74 6.26 5.82 3.26 2.79 7.13

12.25 13.72 9.14 16.55 11.16 7.72

9.16 10.62 10.23 14.92 13.31 8.01

20 6.37 2.00 31.36

19 7.8 l 1.66 21.22

20 4.76 2.75 57.64

19 4.31 2.54 59.03

20 10.77 2.70 25.04

19 10.81 2.31 21.41

12.11 16.60 10.56 12.13

a AUC, area under the curve, normalized to the highest dose; tmax,time to reach maximum plasma concentration; Cmax,maximum plasma concentration; MRT, mean residence time; CV coefficient of variation

Table 4. Coefficients of variation (CV) of the parameters AUC0 t, Cmax, tmax, and total MRT after the first administration and of the intra-individual quotients (value after 2nd treatment divided by value after 1st treatment) a Pharmacokinetic parameter

CV (%)

n

AUC0-t 1st AUC0_t 2nd Quotient

54.4 63.8 26.8

18 18 18

Cma×1st Cmax2nd Quotient

59.4 59.0 34.5

19 19 19

trnax 1st tmax 2nd Quotient

28.7 21.2 30.6

19 19 19

MRT 1st MRT 2nd Quotient

24.4 21.4 28.1

19 19 19

Patient 8 was excluded from the calculation since he died after the first administration

rHulFN-~, [ng/ml] 351

0

4

8

12

16

20

24 Time [h]

a

lated from all individuals irrespective of the dose administered. The time to reach maximum plasma concentration was in the range of 3 . 0 - 9 . 8 h with a mean of 6.4 h (n = 20). The coefficient of variation (CV) was 31.4%. The

Fig. 2. Individual plasma concentration/time curves after the first subcutaneous injection of 0.25 mg/m2 rHuIFN 3, to 7 patients. Each symbol represents one patient

mean total MRT was 10.8 h (n = 20) with a CV of 25.0%. The individual MRT vatues ranged from 6.1 h to 16.6 h. Table 4 shows the CV after the first and second administrations, as well as the CV of the mean quotients. The

173 rHulFN- T [ng/ml] 2.5

1.5

1

\

.5

0

'

0

'

24

'

# 48

72

ù ,~, 144

168

?-; 192 216 Time [h]

Fig. 3. Fitted plasma concentration/time curves (patient 17) after the first and after the second treatment with 0.25 mg/m 2 rHuIFN7

high interindividual variation, as observed for the parameters AUC0-t and Cmax (CV in the order of 60%), is also depicted in Fig. 2: the individual plasma concentration/time curves of the intermediate dose of 0.25 mg/m 2 (first administration) are presented as an example. In contrast, the intra-individual variation of all parameters was low, as the CV of the mean quotients was in the order of only 30%. This is also demonstrated in Fig. 3, where the fitted plasma level/time curves of one patient after the first and the second administration are shown (0.25 mg/m2).

Discussion Subcutaneous administration of interferon was expected to be potentially advantageous in comparison to more conventional i. v. or i. m. administrations in terms of tolerability and possible self-administration for long-term use. Furthermore, it was assumed that the pharmacokinetic properties of extravasal rHulFNy would be altered in comparison to a systemically administered preparation, i.e. plasma concentrations of the lymphokine would be lower but would persist for a longer time. The side-effects associated with rHulFN 7 administration that we observed were similar to those previously reported by Kurzrock et al. [19, 20] e.g. fever and flu-like symptoms (Table 2). However, there was no correlation between the administered dose and the extent of fever induction (Fig. 1). During long-term administration the degree of fever was constant (Fig. 1). This is in contrast to type I IFN, with which patients reportedly develop a tolerance for side-effects like fever and flu-like symptoms [30]. Recent evidence suggests that administration of rHuIFN 7 stimulates release of other lymphokines, such as tumour necrosis factor ~ [22]. It is, therefore, possible that fever was in part associated with a lymphokine induced in vivo. rHulFN 7 as well as any cytokines that were possibly in-

duced did not suppress the counts of granulocytes, lymphocytes, thrombocytes, or erythrocytes after the tested dose during long-term administration. It is possible that during a long-term administration the induction of haemopoetic growth factors [15] outweighs the suppression of haemopoesis [3]. Major objective antitumour responses were observed in only 2 patients. This fact plus the unequal response of lymph nodes in 1 patient, and the regression of lung metastases with simultaneous development of new metastases in another patient all indicate resistance to IFN, which has also been demonstrated in vitro [9]. The plasma level/time curves of rHulFN 7 showed a monoexponential decline suggesting an open one-compartment mathematical model. This plasma concentration profile after s.c. administration is similar to that observed previously for i.m. administration [18]. The absorption process after s.c. administration plays an important role in the kinetic fate of rHuIFN 7. Therefore, the MRT concept is most suitable for the interpretation of our data. The MRTint of rHuIFN 7, as obtained after an intravenous bolus injection, was shown to be in the range of 0.4-1.0 h [19]. The mean total MRT after s.c. administration in our study was 11 h. Since the MRTint contributes only 0 . 4 - 1 h to the total MRT, the MRTabs is in the order of 10 h, which represents about 90% of the total MRT. High values for the MRTabs after i.m. and s.c. administration have been observed for insulin [16], erythropoietin [ 13] and interferon c~ (authors' unpublished results). The mean AUC0-t and Cmax values were shown to be dose-proportional for doses of 0.1 mg/m2, 0.25 mg/m2, and 0.5 mg/m2; however, the interindividual variabilities for these parameters were quite large within each dosage group. In contrast, the intra-individual variance was relatively low. Inter- and intra-individual variance was relatively low for the parameters tmax and MRT (Table 4). The high interindividual variation cannot be due to neutralizing antibodies since all patients were negative in the anti-viral assay. Although the occurrence of binding, not neutralizing antibodies cannot be excluded, no antibody formation upon rHuIFNy administration has been reported in the literature [12]. A variable absorption rate could be another possible source for the interindividual variability. As mentioned above, the MRTabs represents approximately 90% of the total MRT. A variable absorption rate should therefore be reflected in a high interindividual CV of the total MRT. Since, however, the interindividual CV of the total MRT is rather low (Table 4) the possibility of a variable individual absorption rate can be excluded. Since the intra-individual variability is low (Fig. 3), individual differences in efficacy of specific elimination, distribution or metabolic mechanisms (receptor binding, rate of internalization, metabolic processing) might determine the pharmacokinetic fate of rHuIFNy after s.c. administration. The individual differences thus result in a high interindividual variability with respect to Cmaxand AUC0-t. This interpretation might require a process to determine an individual dose. In conclusion we have demonstrated that s.c. administration of rHuIFNy was well tolerated, long-term self administration was possible, and a high interindividual but remarkably low intra-individual variation of plasma levels was detected. Simultaneous in vitro studies (e. g. about the

174 r o l e o f o t h e r l y m p h o k i n e s ) a r e n e c e s s a r y to d e t e r m i n e t h e weak antitumour activity of rHuIFNy in vivo.

Acknowledgements. The skilled technical support of Sibylle Rink, and Inge Brand is gratefully appreciated.

References 1. Blalock JE, Georgiades JA, Langford MP, et al (1980) Purified human immune interferon has more potent anticellular activity than fibroblast or leukocyte interferon. Cell Immunol 49: 3 9 0 - 394 2. Borden EC, Fall LA (1981) Interferons: biochemical, cell growth, inhibitory, and immunological effects. Prog Hemato1350:1-641 3. Broxmeyer HE, Williams DE, Lu L, Cooper S, Anderson SL, Beyer GS, Hoffman R, Rubin BY (1986) The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-T. J Immunol 136: 4487-4495 4. Chan BA, Anicetti RV, Klassen DT, Berthold W, Zahn G, Wert MR, Geier DM, Jones A (1986) A sensitive radioimmunoprecipitation assay for the detection of antibody to recombinant human gamma-interferon: comparison to a bioassay neutralization test. J Interferon Res 6 : 3 1 3 - 3 2 0 5. Duncan DB (1955) Multiple range and multiple F-tests. Biometrics 2:1-42 6. Gray PW, Leung DW, Pennica D, Yelverton E, Najarian R, Simonsen CC, Derynck R, Sherwood RT, Wallace DM, Berger SL, Levinson AD, Goeddel D (1982) Expression of human immune interferon cDNA in E. coli and monkey cells. Nature 295:503-508 7. Gutterman JU, Blumenschein GR, Alexanian R, Yap H-Y, Buzdar AU, Cabanillas F, Hortobagyi GN, Hersh EM, Rasmussen SL, Harmon M, Kramer M, Pestka S (1981) Leukocyte interferon-induced tumor regression in human metastatic breast cancer, multiple myeloma, and malignant lymphoma. Ann Intern Med 93:399 -406 8. Gutterman JU, Fein S, Quesada J, Horning SJ, Levine JF, Alexanian R, Bernhardt L, Kramer M, Spiegel H, Colburn W, Trown P, Merigan T, Dziewanowski Z (1982) Recombinant leukocyte A interferon: pharmacokinetics, single-dose tolerance, and biologic effects in cancer patients. Ann Intern Med 96:549 -556 9. Hannigan GE, Gewert DR, Williams BRG (1984) Characterizafion and regulation of c~-interferon receptor expression in interferon-sensitive and -resistant human lymphoblastoid cells. J Biol Chem 259: 9456-9460 10. Heinzel G (1982) Topfit. In: Bozler G, van Rossum JM (eds) Pharmacokinetics during drug development: data analysis and evaluation techniques. Stuttgart, Fischer, pp 207-208 11. Heron I, Hokland M, Berg K (1978) Enhanced expression of 132-microglobin and HLA antigens on human lymphoid cells by interferon. Proc Natl Acad Sci USA 75:6215 12. Jaffe HS, Chen AB, Kramer S, Sherwin SA (1987) The absence of interferon antibody formation in patients receiving recombinant interferon-gamma. J Biol Response Mod O: 576-580

13. Kamf D (1989) Single dose pharmacokinetics of rhEPO after intravenous and subcutaneous administration. Klin Wochenschr [Suppl] 67: 13-14 14. Kirchner H (1984) Interferon gamma. Prog Clin Biochem Med 1:169 15. Koeffier HP, Gasson J, Ranyard J, Souza L, Shepard M, Munker R (1987) Recombinant human TNFc~ stimulates production of granulocyte colony-stimulating factor. Blood 70:55 - 59 16. Kraegen EW, Chisholm DJ (1985) Pharmacokinetics of insulin. Clin Pharmacokinet 10:303 - 314 17. Kfim M (1980) Towards tumor therapy with interferons: part II. Interferons: in vivo effects. Blood 55: 8 7 5 - 885 18. Kurzrock R, Rosenblum M, Sherwin S, Rios A, Talpaz M, Quesada J, Gutterman J (1985) Pharmacokinetics, single-dose tolerance and biological activity of recombinant gamma interferon in cancer patients. Cancer Res 45: 2866-2872 19. Kurzrock R, Quesada J, Talpaz M, Hersh E, Reuben J, Sherwin S, Gutterman J (1986) Phase I study of multiple dose intramuscularly administered recombinant gamma interferon. J Clin Oncol 4: 1101-1109 20. Merigan TC, Sikora K, Breeden JH, Levy R, Rosenberg SA (1978) Preliminary observations on the effect of human leukocyte interferon in non-Hodgkin's lymphoma. N Engl J Med 299: 1449-1453 21. Nathan CF, Murray HW, Wiebe HW, et al (1983) Identification of interferon-y as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med 158: 670-689 22. Nedwin GE, Svedersky LP, Bringman TS, Palladino MA, Goeddel DV (1985) Effect of interleukin-2, interferon-y, and mitogens on the production of tumor necrosis factors c~ and [3. J Immunol 135: 2495-2497 23. Östlung L, Einhorn S, Robert KH, Juliusson G, Biberfeld P (1986) Chronic B-lymphocytic leukemia cells proliferate and differentiate following exposure to interferon in vitro. Blood 67: 152-159 24. Perussia B, Dayton ET, Lazarus R, et al (1983) Immune interferon and leukocyte-conditioned medium induce normal and leukemic myeloid cells to differentiate along the monocytic pathway. J Exp Med 158:2058-2080 25. Quesada J, Swanson DA, Trindade A, Gutterman JU (1983) Renal cell carcinoma: antitumor effects of leukocyte interferon. Cancer Res 43:940-947 26. Quesada JR, Reuben J, Manning JT, Hersh EM, Gutterman JU (1984) Alpha interferon for induction of remission in hairy-cell leukemia. N Engl J Med 310: 15- 18 27. Rubin BY, Gupta SL (1980) Differential efficacies of human type I and type II interferons as antiviral and antiproliferative agents. Proc Natl Acad Sci USA 77:5928-5932 28. Stewart WEI (ed) (1979) The interferon system. Springer, Berlin Heidelberg New York 29. Talpaz M, McCredie KB, Mavligit GM, Gutterman JU (1983) Leukocyte interferon-induced myeloid cytoreduction in chronic myelogeneous leukemia. Blood 62: 689-692 30. Thompson J, Brady J, Kidd P, Fefer A (1985) Recombinant alpha-2 interferon in the treatment of hairy cell leukemia. Cancer Treat Rep 69:791-793 31. Tsujimoto M, Yip YK, Vilcek J (1986) Interferon-yenhances expression of cellular receptors for tumor necrosis factor. J Immunol 136: 2441 - 2444