ARTHRITIS TREATMENT

Background of the Invention

It is known to employ levamisole, an immuno- modulator as an anti-inflaπunatory agent, e.g., in the treatment of rheumatoid arthritis, thus see Scott,

Annals of the Rheumatic Diseases, Vol. 37 (1978) pages 259-261, Miller, Arthritis and Rheumatism, Vol. 23, January, 1982, pages 10-16, Barada, Arthritis and Rheumatism, Vol. 25, January, 1982, pages 10-16, Huskis- son, The Lancet, February 21, 1976, pages 393-395,

Veys, The' Lancet, April 10, 1976, pages 808-809, Vischer, The Lancet, November 11, 1978, pages 1007-1012. Levami¬ sole in all of these studies was employed with patients maintained throughout the studies on practically fixed doses of other anti-inflammatory drugs. Levamisole chemically is (-) 2, 3,5,6-tetrahydro-6-phenylimidazo- [2,1-b]thiazole.

Inosiplex is an immunomodulating agent. It is the p-acetamidobenzoic acid salt of N,N-dimeth lamino- 2-propanol : inosine complex 3:1 molar ratio. It is available commercially as Isoprinosine, and its manufac- ture and many of its uses are described in Gordon U. S. patent 3,646,007, Gordon ϋ. S. patent- 3,728,450, and Gordon ϋ. S. patent 3,857,940. The entire disclosure of the Gordon patents is hereby incorporated by reference and relied upon.

ybran, J. Rheumatology, Vol. 8:4, 1981, has reported that inosiplex tested favorably in treating rheumatoid arthritis. The patients were stabilized on an optimum antiinflammatory regimen which remained con-

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stant in the study. This work of ybran and co-workers was also reported by Appelboom in a Presentation at the 2nd ^' International Seminar on the treatment of Rheumatic Diseases in Israel November 24-28, 1981. The regimen included the non-steroidal anti-inflammatory drugs indomethacin (100-150 mg) or aspirin (3 grams) . This is shown in a letter of May 5, 1982, from Wybran and Appelboom to the present inventor and also a letter of August 2, 1982, from Dr. Simon of Newport Pharmaceuticals International, Inc., to Dr. Wybran, which letter was acknowledged by Dr. Wybran on August 9, 1982.

Summary of the Invention

It has now been found that inisoplex has anti-inflammatory action, e.g., in the treatment of rheumatoid arthritis, and that this action is not only observed in the absence of indomethacin and aspirin but is even more pronounced in the absence of such drugs.

It has been discovered that Isoprinosine can suppress the development, intensity and persistence of the delayed-type hypersensitivity response when drug administration in immunized animals is initiated after an antigen challenge. When Isoprinosine is initially administered after the antigen challenge but before the development of the delayed-type hypersensitivity response, drug treatment will suppress the development of inflammation. If, after immunization and during a series of further antigen exposures, Isoprinosine is initially administered after the onset of inflam- mation, treatment will suppress its intensity and persistence.

While Isoprinosine has been identified as an immunoregulatory compound acting primarily on T cell function, with regard to inflammatory processes it may also have hitherto unreported effects that are not directly concerned with im unoregula ion. Isoprinosine can suppress nonspecific inflammation, such as that generated by the administration of the pro-inflammatory agent, carrageenan.

Isoprinosine has exerted both anti-allergic and non-specific anti-inflammatory actions in model systems. The intensity of the Isoprinosine effect is similar to the effects observed for two clinically established anti-inflammatory agents: indomethacin and prednisone.

Brief Description of the Drawings

Figure 1 is a graph of the footpaw swelling in Example 1;

Figure 2 is a graph of the footpaw swelling in Example 2;

Figure 3 is a graph of the anti-inflammatory effect in Example 2;

Figure 4. is a graph of the footpaw swelling in Example 3;

Figure 5 is a graph of the footpaw swelling in Example 4; and

Figures 6, 7, 8, and 9 are block charts of comparisons of footpaw swelling with controls, Iso¬ prinosine, indomethacin, indomethacin with Isoprinosine, sodium salicylate, and sodium salicylate with Isoprino¬ sine described in Example 8.

Detailed Description

Example 1

A STUDY OF THE EFFECTS OF ISOPRINOSINE ON FOOTPAD ^' SWELL¬ ING IN MICE CHALLENGED ON DAY 38 AND RECHALLENGED ON DAY 39 WITH HUMAN SERUM ALBIMIN (HSA) FOLLOWING IMMUNIZATION WITH HSA IN COMPLETE FREUND'S ADJUVANT (CFA)

This is an in vivo experiment. Isoprinosine was given in the drinking water at a concentration of 0.25%.

There were employed male CD-I mice, 5 weeks of age at time of primary immunization, obtained from Charles River.

Primary immunization - 0.5 mg HSA in CFA, total volume 0.03 ml/paw, injected subcutaneously. Day 38 and day 39 challenges - 1 mg HSA in normal saline by oral gavage, total volume 0.1 ml.

The methodology was essentially identical to that developed by Dr. John Tew (Immunology 40:425, 1980) in his studies on antigen persistence. Following initial dorsoventral paw diameter measurements by

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Schnelltaster caliper, 22 mice were etherized and immunized in each hind footpad with 0.03 ml of an emulsion of heat-aggregated HSA in CFA, delivering 0.5- mg HSA/paw. On day 38 (challenge day 0), the animals were randomly divided into three treatment groups, as follows:

Control (8 mice; N = 16 paws) Isoprinosine therapy initiated on Day 0 (7 mice; N = 14 paws) Isoprinosine therapy initiated on Day 1 (7 mice; N = 14 paws)

Baseline footpad diameters were measured and the animals were challenged by oral gavage with 1 mg- HSA in 0.1 ml normal saline. On challenge day 1 (experimental day 39) , footpad measurements were taken, after which the animals were again challenged by oral gavage with 1 mg HSA in 0.1 ml normal saline. Treat¬ ments were continued through challenge day 2; footpad measurements were taken daily through challenge day 3. Paw swellings were determined using the experimental day 38 pre-challenge measurement as a baseline.

The results are given in Table 1 and Figure 1, They establish that the development, persistence, and intensity of the delayed-type hypersensitivity response to oral antigen challenge is suppressed by treatment with Isoprinosine, when administered immediately after or one day following antigen challenge.

Histopathologic analysis of the cell types contributing to the inflammatory exudate have revealed these to be exclusively mononuclear cells, largely monocytic; occasional foci of lymphocyte clusters were also found.

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The results of the drug study indicate that Isoprinosine can be useful in the treatment of delayed-type hypersensitivity responses in parts of the body that antigen or antigen fragments reach via the bloodstream (as well a.s by other routes) . Such disease states could include rheumatoid arthritis and a variety of allergic and dermatologic disorders.

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TABLE 1

Fbotpad Swelling (mn)

Day 1 Day 2 Day 3 Sum Days 1-3 Sim Days 2&3 L R L R L R L R L R

Control 1 0.1 0.2 0.4 0.4 0.1 0.7 0.7 0.6 0.5 2 0.1 0.2 0.3 0.2 0.2 0.5 0.6 0.4 0.4 3 0.3 0.3 0.4 0.5 0.1 0.8 0.9 0.5 0.6 4 0.2 0.1 0.2 0.1 -0.1 0.5 0.1 0.3 0.0 5 0.2 0.3 0.5 0.6 0.3 0.9 1.2 0.7 0.9 6 0.1 0.2 0.5 0.4 0.3 0.9 0.9 0.8 0.7 7 0.1 0.1 0.4 0.1 0.1 0.6 0.7 0.5 0.6 8 0.2 0.2 0.3 0.2 0.1 0.7 0.5 0.5 0.3

Average + SE = 0.18 + 0.02 0.37 + 0.04 0.15 + 0.03 0.70 + 0.06 0.52 + 0.05

Isoprinosine 1 0.4 0.0 0.1 0.0 0.2 -0.1 0.7 -0.1 Day 0 Start 2 0.1 0.3 0.1 0.2 0.2 0.3 0.7 3 0.2 0.1 0.2 0.0 0.1 0.6 0.2 I 4 0.2 0.2 0.2 0.3 0.2 0.6 0.7 I 5 0.2 0.1 0.0 0.1 0.1 0.3 0.3 6 0.2 0.3 0.1 0.1 0.1 0.4 0.5 7 0.0 0.0 0.2 0.1 0.0 0.3 0.1

Average + SE = 0.16 + 0.03 0.12 + 0.02 0.11 + 0.02 0.40 + 0.07 by t Test NS P < 0.001 NS P < 0.01

Isoprinosine 1 0.1 0.2 0.3 0.3 0.1 0.1 0.4 Day 1 Start 2 0.2 0.4 0.2 0.3 0.1 0.3 0.3 3 0.2 0.3 0.4 0.4 0.3 0.8 4 0.0 0.1 0.1 0.2 0.1 0.4 5 0.0 0.2 0.4 0.0 0.2 0.2

Example 2

A STUDY OF THE EFFECTS OF ISOPRINOSINE ON FOOTPAD SWELL¬ ING IN MICE CHALLENGED ON DAY 59 AND RECHALLENGED ON DAY 61 WITH E. COLI IN SALINE FOLLOWING IMMUNIZATION WITH E^ COLI IN COMPLETE FREUND'S ADJUVANT (CFA)

This is an in vivo experiment. Isoprinosine was given in the drinking water at a concentration of 0.25%.

There were employed male CD-I mice, 4 weeks of age at the time of primary immunization, obtained from Charles River.

4 Primary immunization - 3 x 10 E. col in

CFA injected subcutaneously in the footpad, total volume 0.03 ml.

4 Day 59 and Day 61 challenges - 3 x 10 E. coli in saline injected subcutaneously in the footpad, total volume 0.03 ml.

The methodology was essentially identical to that developed by Dr. John Tew (Immunology 40:425, 1980) . Following initial dorsoventral paw diameter measurements by Schnelltaster caliper, 23 mice were

'etherized and immunized in each hind footpad with 0.03

4 ml containing 3 x 10 heat-killed E. coli in CFA. On day 59 (challenge day 0) , the mice were measured for baseline paw diameter, etherized and challenged in both

4 hind footpads with 0.03 ml containing 3 x 10 heat- killed E. coli in saline; this same procedure was repeated on day 61 (challenge day 2) . Treatments were as follows:

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Control (8 mice; N = 16 paws) Isoprinosine initiated on Day 0 (8 mice;

N = 16 paws) Isoprinosine initiated on Day 1 (7 mice; N = 14 paws)

The following "blanks" were "immunized" and "challenged" according to the same schedule as the ex¬ perimental animals; these "blank" groups, like the Controls, received only plain drinking water.

Immunization - Saline; Challenge - Saline (6 mice; N = 12 paws) Immunization - CFA; Challenge - Saline

(6 mice; N = 12 paws) Immunization - E. coli in Saline; Challenge- E. coli (5 mice; N = 10 paws)

Immunization - CFA; Challenge - E. coli (6 mice; N = 12 paws)

Treatments were continued through challenge day 4; footpad measurements were taken daily through day 4 and paw swellings were determined.

The results identify two peaks, each of which developed one day following a footpad challenge (chal¬ lenges on days 0 and 2; peaks on days 1 and 3) . When treatment with Isoprinosine was initiated on day 0 immediately following the initial challenge with E. coli in saline, the degree of inflammation is seen to be even less than that resulting from the injection of the saline/saline and other blanks (see Table 3 and Figure 2) , suggesting a non-specific anti-inflammatory effect, as well as a specific anti-allergic anti- inflammatory effect. When Isoprinosine treatment is

initiated on day 1, following the onset of inflamma¬ tion, Isoprinosine is found to exert a significant anti-inflammatory effect as well (see Table 2 and Figure 3) .

In this model, administration of antigen is local (subcutaneous injection in the footpad) and is in relatively high concentrations, in contrast to the study (Example 1) employing the orally administered challenge with HSA (human serum albumin) . The high local concentration of antigen may be associated with the generation by macrophages of suppressor prostag- landin secretion (PGE) . It is of interest that, in Table 4 (a summary of certain other drug studies) , indomethacin is found to suppress the paw inflamma- _ tion generated by oral HSA but not that generated by paw-injected E. coli. Since indomethacin is a prostaglandin synthesis inhibitor, it may be assumed that its absence of effect on the E. coli model re¬ sults from the different pattern of prostaglandins produced. Specifically, in this model the immuno- suppressor actions of macrophage-derived PGE, may have become so significant an output within the immuno¬ regulatory network that the administration of indo¬ methacin, by depressing the generation of this immunosuppressive regulator as well as other pro- inflammatory prostaglandins, may exert a null net effect.

The importance of this observation is that Isoprinosine can specifically be effectively anti- inflammatory when indomethacin is not. Since it is known that about 30-40% of all rheumatoid arthritics do not respond to compounds of the indomethacin type,

such indomethacin-resistant population can be targeted for treatment with Isoprinosine.

The results of this study also suggest the value of using Isoprinosine in the treatment of a panoply of human dermatologic, allergic, and gastro¬ intestinal disorders that are expressions of hyper¬ sensitivity states.

TABLE 2 footpad Swelling (røm)

Day 1 Day 2 Day 3 Day 4 Sum Days 1-4 Sum Days 3&4

L R L R L R L R L R L R

Control - 1 0.2 0.3 0.1 0.0 0.3 0.3 0.3 0.3 0.9 0.9 0.6 0.6

- 2 0.3 0.5 0.1 0.1 0.2 0.4 0.2 0.3 0.8 1.3 0.4 0.7

- 3 0.4 0.2 0.2 0.0 0.4 0.2 0.4 -0.1 1.4 0.3 O . β 0.1

- 4 0.1 0.3 -0.1 0.1 0.2 0.2 0.0 0.1 0.2 0.7 0.2 0.3

- 5 0.2 0.2 0 , 2 0.1 0.3 0.2 0.1 0.1 0.8 0.6 0.4 0 , 3

10 - 6 0.2 0.1 0.0 0.1 0.2 0.2 0.1 0.2 0.5 0.6 0.3 0.4

- 7 0.1 0.2 0.0 -0.1 0.0 0.2 0.1 0.2 0.2 0.5 0.1 0.4

- 8 0.3 0.3 0.0 0.1 0.3 0.3 0.0 0.1 0.6 0.8 0.3 0,4

Average + SE = 0.24 + 0.03 0.06 + 0.02 0.24 + 0.02 0.15 + 0.03 0.69 + 0 ,08 0.39 + 0.05

Isoprinosine - 1 0.0 0.1 -0.1 0.1 -0.2 0.2 -0.1 0.0 -0.4 0.4

15 Day 0 Start - 2 0.2 0.0 0.0 0.0 0.2 0.2 0.2 -0.1 0.6 0.1

- 3 0.1 0, 3 0.0 0.0 0.1 0.0 0.0 0.0 0.2 0.3

- 4 0.1 0.1 0.1 0.2 -0.1 0.0 -0.2 0.0 -0.1 0.3

- 5 0.0 0.0 0.2 0.0 0.1 -0.1 0.0 0.0 0.3 -0.1

- 6 0.0 0.2 -0.1 0.0 0.1 0.2 0.0 0.2 0.0 0.6

20 - 7 -0.1 -0.1 0.0 0.0 0.0 0.0 0.0 0.0 -0 ,1 -0.1

- 8 0.2 0.2 0.1 0.0 0.2 0.0 0.1 -0.2 0.6 0.0 V

Average + SE = 0.08 + 0.03 0.03 + 0.02 0.06 + 0.03 -0.01 + 0.03 0.16 + 0.07 by t Test = P < 0 .001 NS P < 0.001 P < 0. 01 P < 0.001

Isoprinosine - 1 0.3 0.3 0.0 0.1 0.2 0.2 0.2 0.2 0.4 0.4

25 Day 1 Start - 2 0.2 0.3 0.1 0.2 0.1 0.1 0.1 0.1 0.2 0.2

- 3 0.3 0.4 0.0 0.0 -0.1 0.1 0.0 -0.1 -0.1 0.0

- 4 0.2 0.3 0.0 0.1 0.1 0.3 0.1 0.2 0.2 0.5

- 5 0.3 0.3 0.0 0.2 0.3 0.3 0.1 0.2 0.4 0.5

- 6 0.2 0.3 0.2 0.0 0.0 0.2 0.0 0.1 0.0 0.3

30 - 7 0.4 0.5 0.3 0.2 0.0 0.3 0.2 0.3 0.2 0.6 7 + 0.06

TABLE 3 BLANKS Footpad Swelling (mm)

Day I- Day 2 -OJUL- Day k 't-Pny Total a I _. R

Immunize I 0.2 0.1 0.0 0.1 0.1 0.2 0.0 0.0 0.3 0 Saline 2 0.2 0.2 0.1 0.1 0.1 0.2 0.0 0.1 . 0.6

Challenge 3 -0.1 0.0 0.0 0.0 -0.1 0.0 -0.1 -0.1 -0.3 -0.1 Saline k 0.0 0.1 0.1 0.0 0.0 0.2 -0.1 0.1 o.o

5 0.1 0.1 0.0 0.0 0.3 0.2 0.0 0.1 0.1. 0 6 0.1 0.1 0.1 0.0 0.2 0.1 0.1 0.0 o^ 0.2

Average + SO 0.09 + 0.09 0.04 + 0.05 0.13 ±0.11 0.01 ♦ 0.08 0.27 * 0.27

C Immunize I 0.0 0.1 .0 0.0 0.0 0.1 0.1 0.1 0.1 0.3

CFA 2 0.0 0.0 .0 0.1 0.2 0.1 0.0 0.0 0.2 0.2 ϋ) Chal lcnge. 3 0.1 0.1 .0 0.0 0.2 0.) 0.0 0.0 0.3 0.2 .0 0.0 0.0 0.0 0.1 0.0 0.2 0.0

■_-_! Saline ly 0.1 0.0

5 0.2 0.1 .0 0.1 0.2 0.1 0.1 0.1 0.5 0.1* y ω c 6 0.1 0.1 .0 0.1 0.0 0.2 0.0 0.1 0.1 O.S I π Average + SO 0.08 + 0.06 0.03 + 0.05 0.10 + 0.09 0.05 £ 0.05 0.25 +0.16

(!)

Iffmunlze 1 0.1 0.1 0.0 0.0 0.2 0.2 0.0 0.1 0.3 O E. coll 2 0.1 0.2 0.1 0.0 0.3 0.2 0.1 0.1 0.6 0.5 πi In saline 3 0.2 0.1 0.0 0.0 0.2 0.1 0.1 0.0 0.5 0.2

-I Challenge k 0.2 -0.1 0.1 0.1 0.2 0.2 0.2 0.0 0.7 0.2 E. coll 5 0.1 0.0 0.1 0.0 0.2 0.1 0.1 ,5 O

Average .♦ SO 0.10 + 0.09 0.0*» + 0.05 0.21 + 0.06 O.Oβ ± 0.06 0 3 + 0.16

TABLE 4

Significant Inflammation Suppression In Delayed-Type Hypersensitivity

Antloan Challenge Model

Low Antigen/Oral High Antigen/Local

Therapy- HSA _L_. coll

Iscprinosϊnβ Yes Yes

Cytsxan Yes Yes

IndonethaeIn Yes No

Prednϊsen* Ye*

Levaoϊsol* Yes Yes

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Example 3

A STUDY OF THE EFFECT OF ISOPRINOSINE ON FOOTPAD SWELL¬ ING OF MICE FOLLOWING THE INJECTION OF CARREGEENAN

This is an in vivo experiment. Isoprinosine was given at 500 mg/kg by oral gavage 2 hours prior to carrageenan injection, total volume 0.1 ml.

There were employed male and female CD-I mice, 6 weeks of age, born in house.

The inoculum was 80 yg/paw lambda carrageenan, type IV (Sigma Chemical Co. Lot #40F-0388) , made up in saline, total volume 0.03 ml.

Two hours prior to carrageenan injection, the mice received oral gavage of 0.1 ml distilled water or water containing 500 mg/kg Isoprinosine. Two hours later, following initial footpad measurements using a Schnelltaster caliper, 11 male and 10 female mice were etherized and injected subcutaneously in each hind footpad with 80 yg carrageenan. Footpad measurements were taken at 2, 3, and 4 hours post- carrageenan injection, and paw swellings were deter¬ mined.

The results of this first experiment of Isoprinosine effects on carrageenan edema, shown in Tables 5 and 6 and Figure 4, establish that Isoprino- sine exerts a significant anti-inflammatory effect more marked in male than in female mice.

The mice used in this study were exceptionally hyperactive, indicating an endogenous stress state. In a probably related finding, the 3-hour control peak was about half of what was observed in most previous carrageenan studies. Characteristically, it was found that the anti-inflammatory effects were more pronounced when the control peak reaches the typical elevation. Nevertheless, the acute anti-inflammatory effects observed for Isoprinosine are significant and, at 2 hours, are similar to the acute anti-inflammatory effects observed for 5 mg/kg indomethacin (Isoprino¬ sine, 24% reduction in footpad swelling; indomethacin, 36%) .

TABLE 5 Male Mice

Footpad Swelling (mm)

2 Hours 3 Hours n

Control - I 0.3 0.3 0.2 O

- 2 0.3 0Λ 0 0.3

- 3 0.5 0 0

- l» 0.5

0.5 0 0.5 0.5

- 5 0.5 0.6 0.6 0.7

Average + SE . O.te ± 0.03 0 5 + 0.05

Isoprinosine - I 0.5 0.i» 0.6 0.6

- 2 0.3 0.3 0.3 o ^♦ 3 0.3 0.2 0.2

- l« 0.3

0.2 0.3 0 0.3

- 5 0.3 0.3 0.2

- 6 0.3

0.1. " 0.3 O.li 0.1.

Average + SE 0.32 + 0.02 0.37 ± o.ot» by t Test P < C 1.02 NS

TABLE 6 Fema le Mice

Footpad Swe l l ing (mm)

2 Hours Hours ** Hours

_ w

Control - 1 θΛ 0.3 O 0.3 0.3 ^' 0.2

- 2 0.5 0.5 OΛ 0.5 0.2 0.2

- 3 0.5 0.5 0.8 0.7

- k OΛ OΛ 0.3 0.2 0.5 0.2 0.1 0.1

- 5 0Λ 0.5 OΛ 0.3 0.1

Average + SE 0ΛI i 0.03 0Λ7 + 0.06 0.25 ♦ 0.03

Isoprinosine - 1 oΛ 0.5 0.6 o.5 OΛ oΛ

- 2 0.2 0.2 0.3 0.3 0.3 0.1

- 3 OΛ 0.3 OΛ 0.3 0.3 0.2

- *» 0.6 0.3 0.6 0.3 0.3 0.1 I

- 5 0.2 OΛ 0.2 OΛ 0.2 OΛ y oo I

Average ± SE 0.35 ± 0.(Λ 0.39 + O.tΛ 0.27 ♦ 0.0*1

• by t Test NS NS NS

Example 4

FURTHER STUDY OF THE EFFECT OF ISOPRINOSINE ON FOOTPAD SWELLING OF MICE FOLLOWING THE INJECTION OF CARRAGEENAN

This is an in vivo experiment. Isoprinosine was given at 100 and 500 mg/kg by oral gavage 2 hours prior to carrageenan injection, total volume 0.1 ml.

There were employed male CD-I mice, 4 weeks of age, obtained from Charles River.

The inoculum was 80 yg/paw lambda carrageenan, type IV (Sigma Chemical Company Lot #40F-0388) , made up in saline, total volume 0.03 ml.

Two hours prior to carrageenan injection, 24 mice were given an oral gavage of 0.1 ml of distilled water or water containing 100 or 500 mg/kg Isoprino- sine. Two hours later, following initial dorsoventral paw diameter measurements using a Schnelltaster caliper, the mice were etherized and injected sub- cutaneously in each hind footpad with 80 yg carrageenan. Footpad measurements were taken hourly for 4 hours post-carrageenan injection, and paw swellings were determined.

The results, shown in Table 7 and Figure 5, confirm that Isoprinosine suppresses carrageenan- induced footpad swelling. Both the 100 and the 500 mg/kg doses exerted very significant anti-inflammatory effects.

A two-point dose response curve can be gene¬ rated from the data.

The Isoprinosine anti-inflammatory effects that were generated in this study are more profound than in Example 1, and are somewhat more pronounced than typically observed for 5 mg/kg indomethacin.

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TABLE 7 CARRAGEENAN Footpad Swel l ing (mm)

1 Hour 2 Hours 3 Hours 4 Hours

L JL

Control I OΛ 0.5. OΛ 0.5 0.5 0.5 0.3 OΛ 2 0.3 0.3 OΛ oΛ 0.5 0.5 0.3 0.3 3 0Λ 0Λ 0.3 0.6 OΛ oΛ 0.2 0.3 *t 0Λ 0Λ 0.5 0.5 0.5 0.5 0.3 0.2 5 0.3 0.2 0.1) oΛ 0.5 0.6 0.3 0.3 6 0.2 0.3 0.3 oΛ OΛ 0.5 0. 1 0.3

7 0.3 oΛ 0.3 0.5 oΛ 0.6 0.3 0.5 8 0Λ _P__ 5 OΛ OΛ 0.3 0.3

(A Average + SE - O.36 + 0.02 0Λ3 ♦ 0.02 0Λ8 + 0.02 0.29 + 0.02 C CO Isoprino ine I 0.3 OΛ oΛ 0.3 oΛ 0.5 0.2 0.3 100 ng/kg 2 0.2 0.2 0.3 0.3 0.3 0.3 0.2 0.2 3 0.3 0.2 0.2 0.3 0.3 OΛ 0.2 0.2 1

H e <t 0.3 OΛ 0.3 0.3 0.3 oΛ 0.3 0.3 y 5 0.3 OΛ 0.3 oΛ 0.2 0.3 0.2 0. 1 1 pi 6 0.3 0.2 0.3 oΛ 0.3 OΛ 0.3 0.3 7 0.2 0.2 0.2 0.3 0.2 0.2- 0. 1 0.0 8 0. 1 OΛ oΛ Or? OΛ 0.2 0.3

i Average + SE 0.27 + 0.02 0.31 + 0.02 0.33 + 0.02 0.21 ± 0.02 by t Test p < oTbi p <■ 0.001 p < 1 J.OOJ P < 0.02

That Isoprinosine exerts biologically significant anti-inflammatory effects on both immuno¬ logical and non-specific inflammation as pointed out supra is also shown by the following experiments.

Example 5

The three new carrageenan studies summarized in this example were carried out using methods identical to those employed in the carrageenan studies in Example 3. In the data presentation that follows, where only the 3-hour time point is given, it is representative.

Study 1

This study establishes that, while Isoprino¬ sine exerts very significant anti-inflammatory effects in the carrageenan model, Levamisole does not. This difference is in contrast to the observations found by applicant for in vivo antigen-specific immunological inflammation and the observations made by Hadden (Int. J. Immunopharmac. 1 _^ :17-27, 1979) on in vitro immune parameters, especially lymphokine action and PHA- induced lymphocyte proliferation, where both drugs had similar effects.

The effect of Isoprinosine on non-specific inflammation, as demonstrated in the carrageenan model indicates broader anti-inflammatory uses for Isoprino¬ sine than for Levamisole, and to predict an earlier onset of anti-inflammatory effects than that observed for Levamisole in the treatment of rheumatoid

JURI

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^5 ϊPθ

arthritis, wherein both immunological and non-immuno- logical components of inflammation are operative.

Footpad Swelling (mm) at 3-hr Post-Carrageenan % Drug Effect Control 0.53 + 0.04

Isoprinosine

100 mg/kg 0.30 + 0.03* 43%

Isoprinosine

500 mg/kg 0.32 + 0.04* 40% Levamisole

2.5 mg/kg 0.48 + 0.04 9%

Levamisole

-25 mg/kg 0.52 + 0.03 2%

* P < 0.001

Study 2

In the disclosure of Example 4, there is reported a two-point dose response curve for Isoprino¬ sine's anti-inflammatory action in carrageenan edema. The animals used in that study had been shipped to applicant's laboratory on the day preceding their use. Since applicant has found certain parameters of drug response to shift as animals habituate to new housing conditions, another does response study was carried out on additional animals from the same shipment one week later. These results identify an anti-inflammatory response plateau that lies between 50 and 200 mg/kg, with the maximum response locating at the low dose end.

Footpad Swelling (mm) at 3-hr Post-Carrageenan % Drug Effect

Control 0.36 + 0.03

Isoprinosine 50 mg/kg 0.27 + 0.03* 25%

Isoprinosine 100 mg/kg 0.28 + 0.02* 22%

Isoprinosine 200 mg/kg 0.27 + 0.03* 25%

* P < 0.05

Study 3

This study compares effects of Isoprinosine, Indomethacin, and Prednisone on carrageenan edema in the standard system described in the preceding exam- pie. Drug comparison across the 3-hour response point revealed similar anti-inflammatory effects for all the compounds; however, when effects across 1-, 2-, and 3-hours post-carrageenan are summed and com¬ pared, then Prednisone and 100 mg/kg Isoprinosine emerge as the more effective drugs. In this study, both 500 mg/kg Isoprinosine and Indomethacin are observed to exert an early suggestion of a pro- inflammatory effect. A transient pro-inflammatory effect for Indomethacin has been observed by applicant in other carrageenan systems and, interestingly, has been reported clinically as an occasional response observed at the onset of treatment.

The possible significance of this dose effect is that, in studies of Isoprinosine in human inflammatory disease, a specific change in regimen for

an individual non-responder patient may be to lower the drug dose .

Footpad Swelling (inn) Sum

1 Hour 2 Hours 3 Hours Hours 1 - Control 0.29 + 0.03 0.47 + 0.03 0.51 + 0.02 1.26 + 0.06

Isoprinosine .

100 mg/kg 0.26 + 0.03 0.39 + 0.03^ 0.36 + 0.02- 1.01 + 0.07^. Drug effect 10% 17% 29% 20%

500 gAg 0.39 + 0.04°- 0.46 + 0.04 0.32 + 0.03- 1.18 + 0.09 Drug effect -34% 2% 37% 6%

__nd__methacin* 0.36 + 0.03 0.39 + 0.04 0.34 + 0.03^ 1.09 + 0.09 Drug effect -24% 17% ~ ^{33% 13%}

Prednisone** 0.29 + 0.03 0.26 + 0.03^- 0.24 + 0.04- 0.79 + 0.08 - Drug effect 0% 45% 53% 37%

* Indomethacin ό se - 5mgAg; ** Prednisone dose - 2.5 mg/kg a P < 0.001; b P < 0.02; c P < 0.05

Following the intra- footpad inj ection of 500-100 yg lambda carrageenan for each 20 grams of body weight in either mouse or rat , footpad swelling develops progressively over several hours , typically peaking in the neighborhood of 3 hours in the mouse and 4 hours in the rat . The early swelling, as a 1 hour , is due largely to the local release of biogenic amines and bradykinin . It consists principally of a fluid influx resulting from increased vascular per¬ meability . After 2 hours , inflammatory components come principally from the influx of monocytes and the release of lysosomal enzymes , tissue-damaging active oxygens and pro-inflammatory prostaglandins from this

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cellular infiltrate. Pharmaceutical laboratories concerned with screening anti-inflammatory compounds frequently use the 3-hour post-carrageenan time period as a single time,point at which drug actions are assessed and compared. This is the established 3-hour "window" through which one can observe thera- peutically useful anti-inflammatory drug effects.

Example 6

ISOPRINOSINE SUPPRESSION OF CARRAGEENAN FOOTPAD EDEMA IN THE RAT

This is an in vivo experiment. Isoprinosine given by oral gavage 2 hours prior to carrageenan ^• injection, total volume 0.5 ml.

There were employed male Sprague-Dawley rats, 180-200 g, 6-7 weeks of age, obtained from Harla .

The inoculum was 1 mg/paw lambda carrageenan, type IV (Sigma Chemical Col. Lot #40F-0388) , made up in saline, total volume 0.05 ml.

The method classically used in the pharma¬ ceutical industry for the study of drug effects on carrageenan edema was employed in the study of Iso¬ prinosine: the experimental animal was the laboratory rat and the lambda carrageenan thus employed was 1 mg/paw, which produces a diameter increase at 3 hours of about 100%.

Two hours prior to carrageenan injection, the rats received an oral gavage of 0.5 ml distilled water with or without Isoprinosine. Two hours later, following initial footpad measurements using a Schnelltaster caliper, rats were etherized and in¬ jected subcutaneously in each hind footpad with 1 mg carrageenan. Measurements of footpad diameters were carried out at 3 hours, and paw swellings were deter¬ mined.

Part A consisted of two groups of rats:

Control and Isoprinosine, 6 rats/group, N = 12 paws. Isoprinosine.was given at 150 mg/kg.

Part B consisted of four groups of 5 rats _. , N = 10 paws, as follows:

Control

Isoprinosine, 15 mg/kg Isoprinosine, 50 mg/kg Isoprinosine, 150 mg/kg

Part A - Increase in control footpad di¬ ameter at 3 hours was 96.7%.

Footpad Swelling at 3 Hours ^■• mm ~ % DE* by t Test

Control 3.53 + 0.12 Isoprinosine 1.82 + 0.24 48.4% P < 0.001

* Drug effect

art B - Increase in control footpad diameter at 3 hours was 104%.

Footpad Swelling at 3 Hours mm % DE by t Test

Control 3.93 + 0.16

Isoprinosine, 15 3.35 + 0.19 14.8% P < 0.05

Isoprinosine, 50 3.16 + 0.12 19.6% P < 0.001

Isoprinosine, 150 2.56 + 0.26 34.9% P < 0.001

These results show that Isoprinosine sup- presses carrageenan footpad edema in the rat and that this effect is dose dependent between 15 and 150 mg/kg.

These data establish the anti-inflammatory effectiveness of Isoprinosine in a second species, the rat, which is the animal typically used by the industry in screening for anti-inflammatory action via the carrageenan footpad edema model.

The depression in swelling of between 35% and 48% is an excellent drug effect in comparison with that generated by the industry standard, indomethacin. Thus, C.A. Winter of Merck Institute for Therapeutic Research finds indomethacin to suppress carrageenan footpad edema by 32% at 1 mg/kg, 40% at 3 mg/kg, and 50% at about 8 mg/kg (in Non-Steroidal Anti-Inflamma¬ tory Drugs, Excerpta Medical Foundation, New York, 1965, pp. 190-202) . Three mg/kg of indomethacin is the maximum total daily dosage of this anti-inflamma¬ tory drug that is ever given to humans.

Example ^' 7

ISOPRINOSINE SUPPRESSION OF KAOLIN FOOTPAD EDEMA IN

THE RAT

This is an in vivo experiment. Isoprinosine, 150 mg/kg, given by oral gavage 2 hours prior to kaolin injection, total volume 0.5 ml.

Male Sprague-Dawley rats, 200 g, 6 1/2 weeks of age, obtained from Harlan, 6 rats/group (N = 12 paws) .

The inoculum was 10 mg/paw kaolin (hydrated aluminum silicate; Sigma Chemical Co. Lot #101F-0412-) , made up in saline, total volume 0.1 ml.

Kaolin (hydrated aluminum silicate) is frequently used in the pharmaceutical industry to demonstrate the broad spectrum nature of non-specific anti-inflammatory drug effects.

Two hours prior to kaolin injection, rats received an oral gavage of 0.5 ml distilled water with or without Isoprinosine (150 mg/kg) . Two hours later, following initial footpad measurements using a Schnelltaster caliper, rats were etherized and injected subcutaneously in each hind footpad with 10 mg kaolin. Measurements of footpad diameters in this slowly developing inflammation were carried out at 7 hours, as recommended (Wong, S., J. F. Gardocki, and T. P. Pruss, J. Pharmacol. Exp. Therap. 185, 127-138, 1973) .

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Increase in control footpad diameter at 7 hours was 75.5%.

Footpad Swelling at 7 Hours

% Drug t Test mm Effect Statistics

Control 2.78 + 0.18

Isoprinosine 2.18 + 0.15 21.6% P < 0.02

These results illustrate that Isoprinosine can suppress kaolin footpad edema in the rat.

These data confirm that Isoprinosine posses¬ ses a broad spectrum anti-inflammatory action that can operate independent of immune mechanisms.

As regards the modest nature of the drug effect at the dose studied, it should be noted that kaolin edema is typically more resistant than carra- geenin edema to anti-inflammatory action (Garattini, S., et al, p. 151 in International Symposium on Non-Steroidal Anti-Inflammatory Drugs, S. Garattini and M. Dukes eds., Exerpta Medica Foundation, Amster- dam, 1965) .

Example 8

ISOPRINOSINE INTERACTION WITH INDOMETHACIN AND SODIUM SALICYLATE, TWO ESTABLISHED NON-STEROIDAL ANTI-INFLAM¬ MATORY DRUGS

In a study of the treatment of human arthritis as set forth above, Wybran and Appelboom have reported that the addition of Isoprinosine to a regimen of indomethacin or acetylsalicylic acid (aspirin) intensified anti-arthritic effects in 9/15 cases; in 2/15 cases, addition of Isoprinosine made the inflammatory condition worse. It is significant that the Wybran and Appelboom study did not examine Isoprinosine alone for its effects on this infla ma- tory condition, but rather examined Isoprinosine only in combination with known non-steroidal anti-inflamma¬ tory drugs (NSAID) .

The potential for significant drug inter¬ action renders the Wybran and Appelboom data difficult to interpret. These questions come to mind:

Did Isoprinosine act through a prolongation of the half-life or potentiation of the pharmaco¬ logical activity of the other drugs?

Did Isoprinosine, in some individuals, exert an antagonism to the effects of the anti- inflammatory treatments:

In the present studies, experimental inflammation was produced in mice by the means described in the previous example. Immunization was by human serum albinim (HSA) in complete Freund's adjuvant (CFA) per hind paw footpad and antigen challenge enhancing or producing the inflammation was by administration of HSA orally or by footpad injection, as noted, between days 22-43 following immunization. Three separate studies were carried

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out, employing 14-16 paws per treatment group. In this example, the drugs were fed orally.. The dosages employed were as follows:

Isoprinosine — mg/kg Indomethacin — mg/kg Indomethacin mg/kg with Isoprinosine — mg/kg Sodium salicylate mg/kg Sodium salicylate — mg/kg with Isoprinosine — mg/kg

The results shown in Figures 6-9 confirm applicant's observation that Isoprinosine can inhibit inflammation in mammals and depict the mutual inter¬ ference that occurs between Isoprinosine, on the one hand, and indomethacin and sodium salicylate, on the other, when Isoprinosine is used in combination with either of the other drugs.

The previous examples have established that Isoprinosine can exert broad-spectrum anti-inflamma¬ tory drugs. In the studies discussed in this example, it is shown that Isoprinosine and NSAID can signifi¬ cantly inhibit each other's anti-inflammatory actions. Combining drugs can, in fact, result in a significantly poorer anti-inflammatory effect that using Isoprino¬ sine or the other drug alone.

It is possible that this "negative" inter- action derives from the fact that, while NSAID inhibit the synthesis of all pro- and anti-inflammatory prostaglandins, Isoprinosine strikingly potentiates the anti-inflammatory actions of the anti-inflammatory prostaglandin, PGE.. In any event, this kind of interaction may have contributed to the results of Wybran and Appelboom.

The inosiplex of the invention can be used to treat non-specific inflammatory diseases, e.g., rheumatoid arthritis, in mammals such as humans, monkeys, cats, dogs, rats, mice, horses, cattle, sheep, and pigs.

The compositions employed can comprise, consist essentially of, or consist of inosiplex. The inosiplex is usually employed in a pharmaceu¬ tically acceptable carrier, e.g., water, or as a tablet or capsule with appropriate pharmaceutical carriers.

The inosiplex can be administered orally, paranterally, or interparenterally, or by other means.

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