AU660504B2 - Treatment of nephritis - Google Patents

Description

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.9 099~' 90 09 U 0 B* 01, 0 Applicant(s): MONASH MEDICAL CENTRE invention Title: TREATMENT OF NEPHRITIS 84 The following statement is a full description of this invention, including the best method of performing it known to me/us:.

2 TREATMENT OF NEPHRITIS This invention relates to a new method for the treatment of nephritis conditions, in which the active agent used in treatment compositions is deoxyspergualin or an analogue thereof.

Background and Prior Art The antibiotic spergualin was first isolated from culture filtrates of the microorganism Bacillus laterosporus strain BMG 162-aF 2 (Takeuchi et al, 1981).

Its structure has been determined, and the ompound has been totally synthesised (Umezawa et al, 1981; Kondo et al, 1981). Subsequently a derivative of spergualin, 15-deoxyspergualin whose full chemical name is -(15S)-l-amino-19-guanidino-11-hydroxy-4,9,12-triaza- S 15 nonadecane-l0,13-dione (Iwasawa et al, 1982). A number of other derivatives of spergualin, including "15-deoxyspergualin (DSG) which is a racemic compound, have e: been identified and synthesised (Umeda et al, 1985).

Spergualin has been found to have activity not only against a variety of bacteria, but also to be an antitumour agent (Takeuchi et al, 1981). Of the analogues referred to above, DSG has particularly strong anti-tumour activity (Umeda et al, 1985), and has also been found to have immunosuppressive activity in a variety of 25 experimental and clinical situations. This compound is able to suppress tissue graft rejection (Umezawa et al, 1985), and also suppresses adjuvant arthritis and experimental allergic encephalitis in rats (Tabira, 1986) and production of antibody against sheep red blood cells in immunized mice (Ishizuka, 1985); it has been found to inhibit experimental lupus nephropathy in both the BXSB and MRIL/lpr mouse models and the NZB/WF1 mouse model (Makino et al, 1987; Okubo, 1988). In rats with active Heymann stafta/keepWRETYPES128233.92 nephritis, serum antibody production and recognition of nephritis-ralated antigens was inhibited by DSG (Sato et al, 1987). The drug is currently undergoing clinical trials for the treatment of leukaemia and for the prevention of recurrent graft rejection in renal transplant recipients.

In animal studies DSG has been given by the intravenous, intraperitoneal and subcutaneous routes; however, at present it is available for human administration only as an intravenous preparation.

Although it is readily soluble in water, methanol and formic acid, an oral formulation is not available, reportedly due to problems with absorption (Takahashi and Ota, 1991). All of the reported animal and human studies 15 have shown only a few minor side-effects, of which the only one regarded as significant is suppression of the white o blood cell count; however, over a five day treatment regime, this has not caused problems serious enough to justify termination of the treatment.

Glomerulonephritis is the major cause of morbidity and mortality from kidney disease, and this condition is the underlying cause of terminal renal failure in one-third of patients with renal failure requiring dialysis or transplantation. Consequently effective 25 treatment, or better still prevention, of glomerulo- •nephritis would very greatly contribute to public health, as well as reducing the enormous economic costs associated with the maintenance of patients on dialysis and with transplantation.

Nephritis is a term used to describe renal diseases which are inflammatory in nature. These include interstitial nephritis and primary and secondary glomerulonephritis. Primary and secondary glomerulonephritis are the most common underlying causes of .9 9.

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0 B9 B end stage renal disease. Primary glomerulonephritis is understood to be an immunologically based disease causing renal damage by an inflammatory process. Secondary glomerulonephritis is a large group of diseases in which kidney damage is associated with a range of systemic diseases which are predominantly autoimmune in nature. In addition, interstitial nephritis is a less common condition in which inflammation of the kidney is predominantly based in the interstitium. All of these diseases can be induced by a wide variety of agents, including deposition of immune complexes, bacterial or viral infection, autoimmune diseases, some drugs, and some tumours.

Most nephritides are caused by autoimmunity to antigens within the kidney or elsewhere. Exogenous 15 antigens, such as bacterial and viral molecules, can also trigger a nephritic reaction by accumulation of immune complexes within the glomerulus. Morphological and immunochemical classifications have been attempted, but are of limited utility. In particular, animal studies show 20 that the same pathogenetic mechanism, for example deposition of immune complexes involving a single defined antigen, can produce glomerular disease comprising a wide range of functional and histological abnormalities, depending on a variety of factors such as the duration and 25 rate of deposition of complexes in the kidney.

The two major mechanisms of glomerular injury are: a) glomerular localisation of circulating antigen-antibody complexes; referred to as immune complex disease, and recognised by a characteristic pattern of granular deposits containing immunoglobulins and complement in the glomerular capillary walls, and b) glomerular fixation of antibody directed against the glomerular basement membrane, giving rise to typical smooth linear deposition of antibody in a continuous distribution along the glomerular basement membrane.

The first mechanism is non-specific, while the second involves specific antibody directed against a renal component.

The clinical syndromes of glomerulonephritis are known under a variety of names, including acute glomerulonephritis, acute crescentic glomerulonephritis, anti-basement membrane glomerulonephritis, chronic glomerulonephritis, IgA nephropathy, membranoproliferative glomerulonephritis, membranous glomerulonephritis, and mesangial proliferative glomerulonephritis. Other terms will be known to the person skilled in the art. In 15 particular, Goodpasture's syndrome is a severe, rapidly progressive crescentic glomerulonephritis accompanied by intra-alveolar haemorrhage.

Goodpasture's syndrome is a life-threatening disease in which conventional immunosuppressive therapy, usually involving simultaneous administration of prednisolone, cyclosporine, and cyclophosphamide, has had only very limited success, and has major side-effects.

U *o We have now surprisingly found that DSG suppresses both glomerulonephritis and lung haemorrhage in 25 an animal model of Goodpasture's syndrome, without causing any significant side-effects.

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Summary of the Invention According to one aspect of the present invention, there is provided a method of treatment of glomerulonephritis conditions comprising the step of administering to a mammal in need of such treatment a therapeutically effective amount of a spergualin derivative.

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Preferably the spergualin derivative is DSG.

The invention is applicable to the treatment of any immunologically-mediated nephritis condition, including all of those referred to above. In particular, the invention is applicable to the treatment of Goodpasture's syndrome.

While the skilled person will recognise that the dose and regime of administration may be varied for individual patients, it is envisaged that a suitable dose range is 2.5 to 10 mg/kg body weight/day; in the more aggressive forms of nephritis, higher doses may be necessary, and the dose will be limited only by symptoms of bone marrow toxicity. It is envisaged that doses of up to 100 mg/kg body weight/day could be used, with symptoms of bone marrow toxicity counteracted by concomitant treatment 15 with a lymphokine, for example GM-CSF or G-CSF. Anaemia, if encountered, may be treated with erythropoietin.

DSG may be given intravenously or subcutaneously; intravenous treatment may be by continuous infusion for five days or longer depending on response and white blood cell count. Alternatively, DSG may be given by daily three-hour infusions. More than one course of treatment may be necessary, depending upon clinical response.

Oral dosage forms of DSG are not currently approved for human use, but are under investigation, and 25 would be preferred for outpatient treatment.

DSG may optionally be used as part of a multidrug treatment protocol, in conjunction with one or more immunosuppressive agents. The immunosuppressive agent is preferably selected from the group consisting of prednisolone, cyclophosphamide, cyclosporine, and azathioprine.

Other treatment regimes used in the treatment of renal failure, such as antibody therapy with agents such as anti-T cell surface antigens or anti-cytokine antibodies, O S.

Se 5555 Sc cytokine therapy with agents such as GM-CSF or G-CSF, anticoagulant therapy and plasma exchange can be used in conjunction with DSG.

In a further aspect, the invention provides a method of monitoring the response of a subject to therapy, comprising the step of measuring the level of a cytokine in a biological fluid. Preferably the biological fluid is selected from the group consisting ot blood, plasma, serum and urine. Preferably the cytokine is TNFO<, IL-1 or IL-6.

It will be clearly understood that, while the specific description of the invention refers to DSG, spergualin and its other derivatives which are effective against glomerulonephritis and which do not show unacceptable toxicity are within the scope of the 15 invention.

S 4 Detailed Description of the Invention The invention will now be described by way of referene to the followirKn non-i mit-ina =yamn1p. anrl to tho

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5 drawings, in which: Figure 1 shows kidney weight in rats treated with DSG and untreated controls with time after administration of rabbit anti-glomerular basement membrane serum (referred to as NTS); p values were calculated using the ANOVA ttest program; Figure 2 shows glomerular filtration rate, measured as creatinine clearance, in treated and control rats as above; Figure 3 shows proteinuria in treated and control rats as above; Figure 4 shows deposition of rat IgG on the glomerular basement membrane as assessed on 6 pm cryostat sections.

Figure 5 shows the effect of DSG treatment on haematuria in anti-GBM disease. Haematuria was assessed semiquantitatively in untreated animals, and DSG-treated animals. All animals were assessed for haematuria on day 1 and again on the day of sacrifice.

Each point indicates one animal.

Figure 6 shows the effect of DSG treatment on histological changes in anti-GBI disease at day 21, assessed on H&E sections (250X). untreated animal, the enlarged glomerulus exhibited severe hypercellularity, crescent formation segmental necrosis(*) and microthrombosis (arrow). Predominant periglomerular and interstitial infiltration, tubular atrophy and fibrosis .were a striking feature; DSG-treated animal, in 15 contrast to the untreated animal, there is mild glomerular 0 hypercellularity only.

S0 Figure 7 shows that the effect of DSG treatment on leucocytic infiltration in the glomerulus and 0* interstitium in anti-GBM disease at day 21, assessed by immunoperoxidase staining with mAbs. untreated animal: kidney section demonstrating prominent leucocytic infiltration (OX-1 cells) in the glomerulus and 44 interstitium, with focal localisation an area of tubulointerstitial damage. untreated animal: kidney 25 section labelled with the ART-18 mAb, IL-2RA cells were almost exclusively localised to damaged tubulointerstitial a areas. DSG-treated animal: mild to moderate leucocytic infiltration (OX-1+ cells) inglomeruli is apparent with very few leucocytes in the tubulointerstitium; DSGtreated animal: kidney section labelled with the ART-18 mAb, there are no IL-2R cells found. and x250; x320; x125.

Figure 8 shows the effect of DSG treatment on macroscopic pulmonary haemorrhage. Day 14 lung tissue from an untreated animal showing mild pulmonary haemorrhage. Day 14 lung tissue from a DSG-treated animal exhibiting no haemorrhage.

Figure 9 shows the cc.~arison of pulmonary haemorrhage in untreated and DSG-treated animals throughout the experimental course. Each circle represents one animal; closed circles are untreated animals, a.d open circles are DSG-treated. Index of haemorrhage was assessed as described Methods.

Figure 10 shows the effect of DSG treatment on histological changes assessed on H&E sections. Day 7 untreated animal exhibiting mild pulmonary haemorrhage and granulomatous lesions with many foam cells and giant cells present (arrow). Day 21 untreated animal showing 0 aggressive granulomatous lesions and extensive pulmonary fibrosis. Day 7 DSG-treated animal showing little haemorrhage and no granulomatous lesions evident. Day 0 e 0 21 DSG-treated animal showing a normal lung histology (x250).

Figure 11 shows the effect of DSG treatment on spontaneous and LPS-stimulated TNFa production by isolated pulmonary mononuclear cells. Spontaneous TNFa a. 04 production. LPS-stimulated TNFa production. Untreated animals are shown by closed bars and DSG-treated animals by S 25 open bars. Each bar represents a group of 5 animals SEM.

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Untreated and DSG-treated groups were compared by an unpaired t test, p<0.05.

Figure 12 shows the effect of DSG treatment on production of reactive oxygen intermediates (ROI) by isolated pulmonary phagocytes. Spontaneous ROI production. PMA-stimulated ROI production. Untreated animals are shown by closed circles and DSG-treated animals by open circles. Each point represents a group of animals SEM. Untreated and DSG-treated groups were compared by an unpaired t test, p<0.05.

Experimental Model of Goodpasture's Syndrome The experimental model chosen was an experimental glomerulonephritis in the rat induced by treatment with rabbit anti-rat glomerular basement membrane (GBM) serum.

This induces an accelerated anti-GBM disease which is a usefu3 model for the following reasons: this is a severe form of glomerulonephritis with a rapid onset, which exhibits many features of rapidly progressive crescentic glomerulonephritis and Goodpasture's Syndrome in human, and this model progresses to renal failure and a e b* 15 provides the most stringent experimental test for any potential therapeutic agent for the treatment of glomerulonephritis.

*o A detailed description of the experimental model has been published (Lan et al, 1991).

Example 1 -Initial Study of DSG in Experimental Glomerulonephritis *f Rabbit anti-rat GBM serum was prepared by repeated immunization of 4 rabbits with a GBM preparation 25 derived from 500 rat kidneys, over a 4 month period, *seat# according to a standard protocol (Holdsworth et al, 1978).

Accelerated anti-GBM disease was induced in ow o inbred male Sprague-Dawley rats (250-300g) by subcutaneous injection of 5mg normal rabbit IgG, followed 5 days later by intravenous injection of rabbit anti-GEM serum (1ml/100g body weight; day 0).

Preliminary studies with small numbers of animals using 2.5mg DSG with carrier protein (as supplied by the manufacturer)/kg body weight resulted in only minor changes to disease development, such as a reduction of haematuria, but did not alter renal function impairment. Thus, the daily intraperitoneal dose of DSG was increased to which did suppress disease development.

Hence, to evaluate the effect of DSG upon disease induction and progression, the following study was performed: accelerated anti-GBM disease was induced in groups of 5 rats; groups received daily intraperitoneal injections of 5mg/kg DSG or vehicle alone from day 0 until the time of sacrifice. Groups of rats (treated and vehicle only) were sacrificed at days 1, 7, 14, and 21. In all experiments, treated and control animals were age and sex matched.

15 Example 2 Kidney Weight Groups of 5 animals were sacrificed at timed 0 intervals after administration of rabbit anti-GBM serum.

Kidneys were removed, any fluid blotted off, and the tissue weighed. Results are illustrated in Figure 1.

This shows that untreated animals developed an increase in kidney weight above normal, associated with a white inflamed appearance, while DSG-treated animals maintained normal kidney weight and appearance.

On a macroscopic level, the highly significant 25 increase in kidney weight seen in diseased animals (p<0.01) was abrogated in DSG-treated animals, in which kidney r weight remained normal.

9 Example 3 Glomerular filtration rate (gfr) The concentration of creatinine was measured in a sample of serum and in the 24 hr urine collection, both taken at the time of sacrifice. This allows the calculation of the rate of creatinine clearance through the kidneys, which is a measure of the glomerular filtration rate in 12 ml/min. This is a reliable indication renal function.

The results, illustrated in Figure 2, shows that gfr was maintained at normal levels until day 7; there was a significant decrease in gfr at days 14 and 21 in the untreated group while the DSG-treated animals maintained normal gfr.

Impairment of kidney function in this model was prevented by DSG-treatment. This result also indicates that DSG has no overt renal toxicity.

Example 4 Serum Creatinine A significant increase in serum creatinine levels, a standard marker of renal function, developed in untreated animals at day 14, increasing at day 21. In 15 contrast, there was no increase in serum creatinine levels in DSG-treated animals, even at day 21. (Table DSGtreated animals also maintained a normal range of serum urea throughout the experimental course.

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Day Proteinuria Serum Urea Serum Crcacininc Creatinine Clearance Untreated u.oup 1104±59 60:t1.0 49.0:1L.8 I. 29:0.,10 7 265-z40 11.9±1.5 56.83:6.7 130±0.07 14 445:57 29.5±7.1 67.0:5.1 0,89=0.09 2)1 440: 70 51.4:t4.2 92.6± t1 0-52±0.02 DSP-treatcd Pgrou~ 1 39:5 7.6:0O.2 59.2±2.0 1 60=0.08 7 247i-5 0.5±0.9 543:3.0 1.45:0.17 14 19 4 i 62 10.4:1.5c 49.8::4,9' 1, 38 :.0 9 1 2L 308:110 11.5:1.6c 48.8.2.4c 1A4±0.07c Normal Rat 6±1 6.0:0O.2 45.0:t2.0 1.23:0.1 Data are expresscd as mcan of 5 animals SEN4.

a p<0.05, b p<O.O1, cp<0.001, compared to the time-matched untreated group.

*3 f 3 9a Oa. a Oa 9 4 *0e *.a a a a. a Example 5 Proteinuria Animals were placed in cages for 24 hrs with access co water only, and total urine output was collected through the mesh floor of the cage into a cylinder. Total urine volume and protein concentration were determined, producing a measure of urine protein output per 24 hrs.

The results, illustrated in Figure 3, show that all animals developed mild proteinuria (protein output above normal levels) at day 1, which increased by day 7.

In untreated animals, proteinuria became severe at day 14 and 21, while in DSG-treated animals this did not reach such high levels (p<0.05 at day 14). In untreated rats, proteinuria at day 14 was 460 mg/24h, while in treated rats this was reduced to 230 mg/24h.

15 Proteinuria is due primarily to the initial deposition of antibody on the GBM and the subsequent transient polymorphonuclear cell influx. DSG appeared to have no effect on this initial process of glomerular injury, although DSG takes approximately 3 days to take effect, which could be important. DSG was able to reduce the severity of proteinuria, but during the short timecourse of this experiment was unable to resolve it completely.

I 25 Example 6 Haematuria The presence of red blood cells in the urine was seen in almost all animals at day 1, and was possibly I associated with the serum injection. Untreated animals maintained haematuria, which became severe with time (Fig.

5a). In contrast, haematuria was not detectable from day 7 onwards in DSG-treated animals (Fig. Example 7 Immunoglobulin deposition Semiquantitative measurement of rat IgG on the GBM was made on 6Rm cryostat sections. Immunofluorescence labelling with an anti-rat IgG antibody was performed, and the concentration of the antibody titrated. Sections were analysed, and the titre of antibody at which a fluorescence signal was no longer detectable recorded.

The results, illustrated in Figure 4, show that the initial rat IgG deposition was not significantly changed with DSG-treatment, but from day 7 onwards, there was a significant reduction in rat IgG deposition (60-65%; p<0.01). Similar results were seen in alveolar basement membrane, in which IgG deposition was reduced by 75-90%.

Immunofluorescent microscopy revealed linear deposition of rabbit IgG, rat IgG and C3 along the GBM at day 1. The intensity of immune deposits in the glomerulus 15 was semiquantitatively assessed by serial dilution of the S* same antibody with PBS. DSG-treatment made no difference to the deposition of rabbit anti-GBM IgG throughout the experimental course.

In untreated animals, deposition of rat IgG on the GEM increased from day 1 (antibody dilution range 1/200-1/400, mean 1/300) to day 21 (range 1/800-1/600, mean 1/1040). Compared to untreated animals, DSG-treatment made no difference to rat IgG deposition at days 1 and 7, but it did significantly suppress deposition at day 14 (range 25 1/200-1/800, mean 1/360) and day 21 (range 1/200-1/800, mean 1/380). A similar suppression of glomerular deposition of rat C3 was apparent in DSG-treated animals.

In addition, glomerula fibrinogen deposition was apparent at day 1 in untreated animals and increased with time.

Fibrinogen deposition was significantly suppressed at all time points by DSG-treatment. Statistical analysis showed that there was a significant difference between DSG-treated and untreated animals in the deposition of rat IgG, C3, and fibrinogen in the glomerulus over the experimental course (p<0.001 by ANOVA).

The reduced level of rat IgG deposition implies that there is a lower level of immunoglobulin production by the DSG-treated animals. Thus, DSG appears to be suppressing some stage(s) in B cell IgG production, which is important in many kidney diseases where continued immune-complex deposition occurs. Since the deposition of rabbit IgG on the GBM (the target for rat IgG deposition) was unaffected by DSG-treatment, it appears that a reduction in rat IgG production is involved.

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To evaluate the degree of glomerular damage, the percentage of glomeruli with lesions of atrophy, fibrinoid exudation and necrosis, glomerular crescent formation and sclerosis was assessed by examination of at least 100 glomeruli per animal on haematoxylin and eosin- stained sections. The degree of glomerular hypercellularity was also assessed on the basis of total glomerular cell count/glomerular cross-section (gcs) and ranked as described (Lan et al, 1991 normal (less than cells/gcs); slight increase in cell numbers (50-70 cells/gcs); mild (70-90 cells/gcs); moderate 120 cells/gcs); severe hypercellularity (more than 120 cells/gcs).

Tubulointerstitial lesions, such as tubular necrosis and atrophy, interstitial infiltration and fibrosis, were semiquantitatively analysed on haematoxylin and eosin-stained sections and graded on a scale of 0 to 3 as previously described (Lan et al, 1991 with modification: no apparent damage; minor damage interstitial infiltration restricted to hilar and periglomerular areas, tubular atrophy and fibrosis involving less than 10% of the cortex, little tubular necrosis; mild damage half the cortex with leucocytic infiltration, tubular atrophy and fibrosis involving 10-25% of the cortex, less than 5% of tubular necrosis; moderate damage diffuse interstitial infiltration, tubular atrophy and fibrosis involving 40% of the cortex, 5-15% of tubular necrosis; severe damage prominent interstitial infiltration with focal accumulation in the areas of damage, tubular atrophy and fibrosis involving more than 40% of the cortex, more than 15% of tubular necrosis. In addition, the number of 15 tubular protein and/or granular casts was also assessed in 10 lower power fields (10x) and expressed as casts/low power field.

The effects of DSG on the histopathological a* o changes in glomeruli and the interstitium were semiquantitatively assessed in haematoxyl in and eosin-st&ined sections and summarised in Table 2.

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All these changes were evident at day 7 and became severe by days 14 and 21 (Figure 6a, Table In marked contrast, DSG-treated animals exhibited only minor glomerular lesions throughout the experimental course (Figure 6b, Table 2), including an almost complete suppression of crescent formation.

In the interstitium, all untreated animals developed prominent interstitial leucocytic infiltration, moderate to severe focal tubular necrosis and/or atrophy, and numerous protein or red blood cell casts in tubular 15 luminal spaces (2-11 casts/low power field) from day 7 onwards (Figure 6a). A significant interstitial fibrosis was also evident at day 14 and became extensive by day 21.

Again there was a very marked contrast apparent with DSG-treatment. Treated animals showed only mild interstitial infiltration, few protein casts (0.5-2 casts/low power field), and occasionally, very mild tubular necrosis or atrophy from day 7 onwards (Fig 6b). Mild interstitial fibrosis was only apparent in some animals at day 21.

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050 ft ft ft ft 25 Linear regression correlation analysis showed 0 that all the indices of histopathological injury correlated 6 significantly with proteinuria, increased serum creatinine a and decreased creatinine clearance in untreated animals (p<0.001). However, this was not the case in the DSG-treated animals.

Example 9 Glomerular and interstitial leucocytic infiltration In untreated animals, the evolving pattern of leucocytic infiltration followed that previously described in this model (Lan et al, 1991 At day 1, leucocyte infiltration was restricted to glomeruli and the hilar region, with widespread tubulointerstitial infiltration first apparent at day 7. Leucocytes continued to accumulate within both kidney compartments during the experimental course (Figure 7a). The presence of immune-activated T cells within the interstitium was identified by expression of the interleukin-2 receptor (IL-2R) (Lan et al 1991 b; Paterson et al 1987).

Expression of MHC class II I-E antigens was also evaluated as a marker of leucocyte activation.

DSG-treatment resulted in a dramatic suppression of leucocyte infiltration and activation (Figure 7c).

However, there was a differential effect of DSG on leukocytic infiltration in the glomerulus compared to the interstitium. Quantitation of the effect of DSG-treatment on the infiltration of leucocyte subpopulations in both the glomerulus and interstitium is shown in Tables 3 and 4 and described below.

Monoclonal antibodies (mAbs) used in this study were as follows: OX-1, leucocyte common antigen 6 (Sunderland et al, 1979); ED1, most macrophages and some 5 dendritic cells (Dijkstra et al, 1985); OX-42, most 25 macropLages and polymorphs, some dendritic cells (Robinson et al, 1986); OX-19, CD5 antigen, pan T cells (Dallman et al, 1984); W3/25, CD4 antigen, T helper cells, some macrophages and dendritic cells (Williams et al 1977; Steiniger et al 1984); OX-8, CD8 antigen, T cytotoxic/suppressor and natural killer cells (Brideau et al, 1980); OX-17, MHC class II antigen (I-E epitope RT1D) (Fukimoto et al, 1982) (inbred Sprague-Dawley rats do not express the MHC class II I-A epitope recognised by the OX- 6 mAb); ART-18, p55 chain of the rat interleukin-2 receptor (IL-2R) (Osawa and Diamanstein, 1983).

In untreated disease, there was a significant infiltration of leucocytes into the glomerulus (p<0.001 vs.

normal). This glomerular infiltrate was composed primarily of EDI positive macrophages and continued to accumulate throughout the experimental course (Table Associated with glomerular macrophage infiltration was the accumulation of significant numbers of MHC class II I-E" cells. These I-E* cells may represent a subset of stimulated macrophages, or they could be activated resident glomerular cells such as mesangial cells. DSG-treatment significantly reduced, but did not abrogate glomerular macrophage infiltration (Table However, a striking result was that DSG-treatment markedly suppressed the S 15 accumulation of I-E cells within the glomerulus from day 7

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onwards. Compared to normal animals, there was no significant glomerular infiltrate of total T cells (OXindividual T cell subsets (OX-8 or W3/25+), or immune-activated T cells (IL-2R in either untreated or DSG-treated animals.

The glomerular macrophage infiltrate is labelled by both ED1 and OX-42 mAbs, however, it hsq previously been •o demonstrated that heterogeneity of macrophage antigen expression exists in the renal interstitium and different 25 subpopulations of macrophages are labelled by the ED1 or OX-42 mAbs (Lan et al, 1991 Therefore, the number of interstitial macrophages was counted on the basis of dual labelling by ED1 and OX-42 mAbs since no polymorphs were found in the interstitial area.

In untreated animals, there was a marked tubulointerstitial leucocytic infiltration from day 7 onwards (Figure 7a). In contrast to the glomerular infiltrate, the tubulointerstitial infiltrate was composed of both macrophages and T cells. Many of the infiltrating 00 0

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T cells were activated as demonstrated by significant numbers of IL-2R' cells (Figure 7b). There was also a significant increase in the number of OX-8 cells (CD8 T cells and natural killer cells) in the interstitium at day 1 (49+11 cells/mm 2 which increased to 126+36 cells/mm 2 at day 14 and maintained at this level at day 21.

Accumulation of I-E cells followed the pattern of the total leucocyte infiltrate, suggesting immune stimulation of the macrophage, T cell, dendritic and natural killer cell populations.

DSG-treatment dramatically suppressed the tubulointerstitial leucocyte infiltrate (Table 4 and Figure 7c). Compared to normal animals, the total interstitial leukocytic infiltrate (OX-1* cells) was significantly 15 increased only at day 21 The number of ED1/OX- 42' macrophages and OX-19 4 total T cells was not significantly different to that of normal animals throughout the experimental course. In addition, there was an almost complete abrogation of IL-2R expression within the interstitium, even though some T cells were present (Figure 7d). The initial infiltrate of OX-8 cells at day 1 seen in untreated animals was not altered by DSGtreatment (55+12 cells/mm 2 but it did not increase further (day 21 63+8 cells/mm 2 and was 3ignificantly different from untreated animals The number of I-E cells within the interstitium was also reduced by DSG-treatment compared to untreated animals (Table but there was still significant expression above normal This may reflect I-E expression by dendritic cells and possibly by natural killer cells.

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Tible 3. Quantitation of lecocyte subsets in glomeruli Day OX-i EI- 1 OX-19 OX-17 ART-I8 Untreated -rou~ I 9.7t1.1 7.3±0.,4 0.52±0.11 3.6:0.78 0.40!0.40 7 10.3±2.3 11.2±1.5 0.26±0.15 3.8:t1.26 0.26-0.21 14 14.0!1.1 12.6±1.0 0.14±0.09 6.1:0.84 0.32:0.22 21 17.6~ 17.1±2.8 0.001-0.00 5.7:0O.76 0,16i-0,05 0 DSP-trcated giroup *1 6,6:1.2 3.7±1.0 0.42±0.15 4. :0.93 0. 12:0o.09 6.5: 1.2 5.5: 1.31 0.30±0.13 l.1±0 32- 0.12:=0,09 14 7.4 -1.O0a .3t1.4 0.20:0.07 0. 9± 0,33 C 0.68:0.38 21 9.7±2.8' 9 .2± 2 I 0.14±0.06 1.9-~0.49r 0. 14:=0.1t1 *.-Normal rat 0.8:0.1 0.3±0.1L 1.1±0o.05 0.3:0.1t Data are expressed as positive cells per glomerular cross section glomcruli were scored for each animal. Each number represents the mean count of 5 animals per group SEM.

Ip<0.05, b p<0.01, compared to (lhe time-matched untreated group 0 0* *S S P

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S. S Table 4. Quantitation of leucocyte subsets in the tiibulointerstitial area Day OX-i EDI/OX-42 OX-19 O 17 A RT-IS8 Untreated group I 265:t45 83:t18 63t±14 147±30 6:2 7 525±101 280±87 133t24 349±21 78:54 14 753±134 398±58 107:-30 45 1 32 65:215 2)1 676-75 335-114 '2)4:36 655:-71 66:-28 DSP--tre~ated 2,roup 1 163 i14 63:6 55:14 175:27 2= 7 317±66 145=42 83± -3 3 23 1 51 1:1=2 14 296:77c 62i18' 43i15 150i29'c:1 21 394i1052 8t 63: 9 234.:34c It1P Normal rat 102±8 56=5 30±7 53: 10 0:0 Data arc expressed as positive ells per square millimeter, 20 high power fields were countcd per animal. Each number represents the mean of 5 animals SEM.

Ap<o.os, 'p<0.01, c p<O.OO1, compared to the timc-matchzd untreated croup.

Example 10 Pulmonary Haemorrhage and Histological Damage Pulmonary haemorrhage and haemoptysis is evident in approximately two thirds of patients with Goodpasture's syndrome. ExperimentLi Goodpasture's syndrome was induced in male inbred Sprague-Dawley rats using passive accelerated anti-GBM glomerulonephritis a disease model which features both crescentic glomerulonephritis and lung haemorrhage as previously described (Lan et al, 1991 a; Lan et al 1991 In untreated animals, macroscopic pulmonary haemorrhage was clearly evident at day 1 and remained a prominent feature throughout the experimental course (Figs. 8a and Microscopically, untreated animals at 9 day 1 exhibited pulmonary fibrin deposition and oedema, multi-focal or patchy intra-alveolar/broncheal haemorrhage, and prominent perivascular mononuclear cell infiltration.

*0 These lesions became more pronounced at day 7, with all S*i untreated animals exhibiting widespread lung haemorrhage and accumulation of macrophages which phagocytosed many erythrocytes and fibrin-fragments (Fig 10a). In particular, focal granulomatous lesions containing numerous foam cells and giant cells were L.:en in one animal at day 7 (Fig. 10a). Focal granulomatous destruction became more aggressive with time, being evident in all animals at day 25 14 (mean index score 1.8) and day 21 (mean index score 0 In addition, extensive fibrosis was evident by day 21 (Fig. At day 1, DSG-treated animals exhibited very similar haemorrhage and histological damage as that seen i' untreated animals. However, by day 7 of DSG treatment a quite different picture emerged. There was a dramatic reduction of macroscopic haemorrhage with complete resolution by day 14 (Figs. 8b and Microscopically, pulmonary haemorrhage and inflammatory exudation and infiltration was markedly suppressed at day 7 of DSG treatment, and granulomatous lesion formation was not evident (Fig 10c). By day 21, DSG-treated animals had recovered a normal histological structure (Fig. In the kidney, DSG treatment also had a significant effect (Lan et al, submitted for publication).

Initial proteinuria at day 1 was unaffected by DSG treatment, but the degree of proteinuria was significantly reduced from day 7 onwards and renal function impairment was prevented. Microscopically, DSG treatment markedly suppressed histological damage including glomerular necrosis, crescent formation, and interstitial fibrosis (Lan et al, submitted for publication).

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15 Example 11 Immunoglobulin Deposition The effect of DSG treatment on the humoral immune response was assessed. The intensity of immune deposits within the lung was semiquantitatively assessed by direct immunofluorescence on tissue sections using serial dilutions of the detecting antibody. In untreated animals, there was intense linear deposition of rabbit IgG along the ABM throughout the experimental course. Deposition of rat IgG along the ABM was seen in some animals at day 1 rats: mean titre 1/170). At day 7 all animals exhibited linear deposition of rat IgG which increased in intensity with time (mean titre 1/960 at day 21). A similar pattern of C3 deposition along the ABM was evident with 1/5 animals positive at day 1 and all animals positive from day 7 onwards. Fibrinogen deposition was also evident in animals at day 1 (mean titre 1/150) which became more intense with time.

In DSG-treated animals, there was strong linear deposition of rabbit IgG along the ABM throughout the experimental course, very similar to that seen in untreated animals. Deposition of rat IgG was evident in animFls at day 1 (mean titre 1/57). Although almost all animals from day 7 onwards demonstrated rat IgG deposition along the ABM, the titre did not increase significantly (mean titre at day 21 1/120).

Example 12 C3 deposition on basement membranes Deposition of C3 upon both glomerular and alveolar basement membranes (GBM ABM respectively) was semiquantitatively assessed by means of titration of the detecting antibody. Deposition of rat C3 on both the GBM and ABM was suppressed by 80-85% and 70-85% respectively in DSG-treated animals. C3 deposition is dependent upon rat IgG, therefore these results indicate that DSG may be p 15 suppressing B-cell immunoglobulin production.

Deposition of C3 in DSG-treated animals followed a pattern similar to that seen for rat IgG. There was no S" fibrinogen deposition in DSG-treated rats at day 1 and only weak deposition was apparent in 10/15 animals at later times. One way analysis of variance (ANOVA) found that i'v, there was a significant difference in pulmonary deposition of rat IgG, C3, and fibrinogen between DSG-treated and untreated animals over the experimental course used (p<0.001), but the intensity of rabbit IgG deposition was 25 not significantly different between the two groups.

In the kidney, DSG treatment was also found to significantly inhibit the deposition of rat IgG, C3 and fibrinogen, while deposition of rabbit IgG was unaffected (Lan et al, submitted for publication). Thus DSG treatment suppressed the systemic humoral immune response to the administered antigen even though the animals were primed to the antigen. This is consistent with previous studies in which DSG was found to inhibit allograft rejection due to secondary antibody responses (Valdivia et al, 1990).

Example 13 Leucocytic Infiltration Infiltration of leucocytes into the lung was quantitated on tissue sections labelled with mAbs and the results are shown in Table 5. The evolving pattern of leucocytic infiltration in untreated animals followed that previously described in this model (Lan et al, 1991 a).

The majority of infiltrating leucocytes were ED1 macrophages which were diffusely spread throughout the tissue, with intense focal accumulation in haemorrhagic granulomatous lesions. A significant infiltration of R73 T cells was evident from day 7 onwards (p<0.05 vs normal).

The majority of T cells were localized in granulomatous 04 lesions and in perivascular regions. Most infiltrating T cells were of the CD4+ T helper phenotype since the number S 15 and distribution of OX-8+ cells was unchanged over the time course while increased numbers of W3/25' (CD4') cells were evident from day 7 onwards. Of particular interest was the significant increase in the number of cells expressing MHC class II antigens (OX-17+ cells) and the interleukin-2 receptor (IL-2R) apparent from day 7 onwards (p<0.001 vs normal). Both IL-2R+ and OX-17+ cells were primarily localized in perivascular areas and granulomatous lesions.

Linear regression correlation analysis was used to compare semiquantitative results of microscopic pulmonary 4 25 haemorrhage or granulomatous lesion formation with 4 leucocyte infiltration in untreated animals throughout the experimental course. ED1+ macrophage infiltration correlated significantly with pulmonary haemorrhage (r=0.58, p<0.01). T cell infiltration correlated significantly with granulomatous lesion formation (r=0.48, p<0.05), but not with pulmonary haemorrhage. Similarly, the appearance of both IL-2R cells correlated significantly with granulomatous lesion formation (r=0.58, p<0.01).

DSG treated animals also exhibited a significant infiltrate of ED1' macrophages at day 1 (p<0.05 vs normal), but this was reduced compared to the untreated group (Table There was marked suppression of pulmonary leucocyte accumulation in DSG-treated animals from day 7 onwards.

Numbers of ED1 macrophages were reduced to that of normal animals by day 14. DSG treatment also reduced the T cell infiltrate, and markedly suppressed the appearance of IL- 2R cells and cells expressing MHC class II antigens.

Numbers of OX-8* cells were unaffected by DSG-treatment, while W3/25' cells were significantly reduced. Similar results were found in the kidney, where DSG treatment significantly suppressed both macrophage infiltration and the appearance of immune-activated mononuclear cells (Lan et al, submitted for publication). These results are S S 15 consistent vith previous studies showing that DSG inhibited leucocyte infiltration and activation in the prevention of renal allograft rejection (11,14).

*4 0@ p. 4 *r *4 p .p p p 4S* pu Table 5 EfTcct of DSP trcatmncnt on pulmonary leukocyte inFiltration.

Day OX-I EDI R73 OX-17 A RT- 18 Untreated discase 1740:178 875±117 336±53 77817 70i5 7 1883i273 915:163 504±86 1704i271 108:19 36 1- 1680±201 723±85 484 ±44 1727: 228 178.18 1 1521117 802±58 524:80 1401:135 200-.25 DSI'-rcatcd discasc 1 11443:79 4 3 9 7 8 b 311:30 807:50 1511 7 1121:L54' 503±31a 449-70 907i6g' 56:21 14 7 80 6 1 b 263±25c 358±38 8 6 3 :576 6 :l t 21 1067.112 a 361.31c 436±54 1011t131 08:16c #9 Nornmal animals a 745:28 145±9 276±16 540:29 7:1 0.60 Data arc cxprcssd as positive cells pcr square rnilimctcr (rncan SEIM) bp<001t, C p<0.001, compared to thc tirc-matched untrcatcd group Example 14 Tumour necrosis alpha (TNFa) production by pulmonary leucocytes One possible mechanism by which DSG could act is the suppression of macrophage activation. Production of the pro-inflammatory cytokine TNFa is an important function of stimulated macrophages. Thus, both spontaneous and LPSinduced TNFa production by cultured leucocytes cells/ml) obtained from either peripheral blood or isolated from lung tissue was measured using the L929 fibroblast lysis assay linked to an ELISA plate reader.

In untreated animals, spontaneous TNFa secretion by cultured lung mononuclear leucocytes was evident from day 1 to day 21 (Fig. lla). Lipopolysaccharide (LPS) stimulated TNFa secretion (maximal inducible production) 15 was approximately six times greater than spontaneous production (Fig. lib). DSG treatment caused an increase in 4. spontaneous TNFa secretion at day 1, but thereafter it was markedly suppressed, being undetectable in 5/15 animals over days 7-21 (Fig. lla). However, DSG treatment did not S, 20 suppress LPS-induced TNFa production by pulmonary mononuclear leucocytes (Fig. llb).

99 Example 15 Superoxide radical production by pulmonary leucocytes Production of superoxide radicals is another means by which stimulated macrophages can cause tissue t" damage. We employed a sensitive assay in which superoxide radical production results in the oxidation of a rhodamine compound, converting it to a fluorescent molecule detectable by flow cytometry. Thus, the percentage of superoxide producing cells, showing both spontaneous production and phorbol myristate acetate (PMA)-inducible (maximal) production, can be determined.

In untreated animals, spontaneous production of ROI was evident in approximately 40% of cultured pulmonary phagocytes throughout the experimental course (Fig. 12a).

This was increased to appronimately 55% of pulmonary phagocytes following stimulation with PMA (Fig. lib). DSG treatment made no difference to the percentage of cells spontaneous producing ROI except for a significant reduction at day 14 (Fig. lla). DSG treatment caused a modest inhibition in the percentage of cells producing ROI following PMA-stimulated over the experimental course (p<0.05 by ANOVA) (Fig. llb). In positive cells, there was a wide range of intensity of ROI production (measured by o fluorescence intensity), and this was not altered by DSG treatment.

Conclusion Mechanism of Suppression *o 0* Deoxyspergualin-treatment resulted in a ic su 4 re i4 n cF r! <e 4 aa nI o no A aoaa f rf al d isea'se4 Com aredn. O wi L 0 th 0* 0 01 S* .0 4 .000• *0 9 saline treated controls, deoxyspergualin-treatment reduced proteinuria, resolved haematuria, and completely prevented a fall in creatinine clearance. Deposition of rabbit IgG along the GBM was unaffected by deoxyspergualin treatment, but glomerular deposition of rat IgG and C3 was significantly reduced from day 14 onwards. Deoxyspergualin 25 treatment also produced a dramatic improvement in renal histology. Glomerular necrosis, fibrosis, and crescent formation was markedly suppressed while tubulointerstitial lesions were completely prevented. This was associated with a marked suppression of mononuclear cell infiltration and activation. In the glomerulus, macrophage infiltration was suppressed by approximately 50%, whereas accumulation of macrophages and immune-activated (IL-2R*) T cells within the interstitium was almost completely abrogated by deoxyspergualin treatment.

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0e The primary mechanism by which DSG inhibited anti-GEM disease in primed animals was through suppression of leucocyte infiltration and activation within the kidney.

This is consistent with previous studies in which DSG suppressed leucocyte infiltration in the prevention of allograft rejection and in the reversal of acute rejection (Lan et al, 1991 a; Fujii et al, 1990). In the glomerulus, there was only partial inhibition of macrophage infiltration which may explain why urinary protein excretion was only partially suppressed and minor glomerular lesions such as mesangial hypercellularity were still apparent. However, the almost complete suppression of cell-mediated immunity within the interstitium explains the ability of DSG to prevent renal function impairment and 15 damage to the tubulointerstitium.

In addition to suppression of the local kidney cell-mediated immune response, DSG also acted systemically as evidenced by the suppression of rat immunoglobulin deposition in the glomerulus apparent at day 14. Although this is not likely to play a significant role in disease suppression during this 21 day experimental course, it could be very important for the longer term suppression of disease progression since in this primed model there is prominent activation of both T and B cell compartments in peripheral lymphoid tissue which is probably very important in amplifying the immune response within the kidney (Lan et al, Submitted for publication). This suppression of rat immunoglobulin deposition is consistent with studies in which DSG has been shown to suppress progressive splenomegaly, production of anti-DNA antibodies, circulating immune complexes, and glomerular IgG and C3 deposition in spontaneous autoimmune lupus nephritis in susceptible mouse strains (Okubo et al 1988; Ito et al, 1990). DSG was effective in suppressing both 4.

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*6 a 6 64 6r 4 the development of renal injury and reversing established renal injury in this model. Although this spontaneous model of murine lupus is quite different to accelerated anti-GBM disease, it will be interesting to determine whether DSG has similar effects upon glomerular and interstitial leucocyte infiltration and activation in the lupus model.

One intriguing question relating to the action of DSG in anti-GEM disease is the differential effect upon leucocyte infiltration in the glomerulus and the interstitium. This result suggests that there are two different mechanisms of leucocyte infiltration operating in the two compartments. Deposition of rabbit IgG was restricted to the GBM and absent from the interstitium.

15 Hence, glomerular macrophage infiltration may be Fc-receptor mediated, but interstitial leucocyte accumulation may work through other mechanisms (Atkins et al, 1991). Cytokines produced within the glomerulus may diffuse down the mesangial stalk into the interstitium and so attract leucocytes into the periglomerular area which then become wide spread (Atkins et al, 1991). This may in part be facilitated by cytokine induced expression of leucocyte adhesion molecules (Atkins et al, 1991). There is little known of DSG effects upon cytokine production, but one study found inhibition of IL-1 production during the early phase of rat allograft responses (Waaga et al, 1990). However, we have found that DSG is able to suppress interferon-gamma induced upregulation of ICAM-1 expression by monocytes in vitro (unpublished observations) suggesting that DSG may interfere with the mechanism of monocyte migration into the kidney.

This study has demonstrated that DSG is able to suppress the development of rapidly progressive crescentic glomerulonephritis in antigen primed animals. It appears to act by suppressing both the local kidney cell-mediated immune response and acting systemically to reduce immunoglobulin production and to reverse steroid-resistant acute rejection, in the absence of nephtrotoxic effects.

DSG treatment also resolved pulmonary haemorrhage, prevented the appearance of granulomatous lesions and resulted in a histologically normal lung structure by day 21. This improvement was associated with a marked suppression of macrophage infiltration (p<0.001 vs. untreated) and accumulation of immune activated (IL- 2R mononuclear cells (p<0.01 vs. untreated). DSG treatment also suppressed TNFO production but exhibited only a partial suppressive effect upon production of reactive oxygen intermediates (ROI) by pulmonary phagocytes. Deposi- 15 tion of rabbit IgG along the alveolar basement membrane (ABM) was unaffected by DSG treatment, but deposition of rat IgG, C3, and fibrinogen was significantly reduced (p<0.05 vs. untreated).

DSG suppressed pulmonary injury in accelerated anti-GBM disease by acting on the local cellular immune response and the systemic humoral immune response In a

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0 a particular, suppression of TNFa production may be a key mechanism in the suppression of pulmonary injury.

DSG treatment begun at the time of administration 25 of nephrotoxic serum in antigen-primed rats resolved ini:tial pulmonary haemorrhage, prevented granulomatous les.on formation and fibrosis, and facilitated recovery of normal tissue structure. The mechanisms by which DSG suppressed pulmonary injury are discussed below.

We have previously shown that induction of pulmonary haemorrhage in accelerated anti-GBM disease is associated with a transient influx of polymorphonuclear 1' cells, while progression of pulmonary haemorrhage correlated with macrophage infiltration (Lan et al, 1991 The present study confirmed this finding and in addition found that focal accumulation of macrophages, T cells and immune-activated (IL-2R') cells was associated with the development and extent of granulomatous lesions.

These results are consistent with findings in other models of lung injury. Pulmonary injury occurring within 4 hours of administration of anti-ABM antibody in the rabbit is leucocyte dependent (Boyce et al, 1991). In a rat model of pulmonary granulomatosis and in he-an sarcoidosis, macrophages and IL-2R' cells were found in gianulomatous lesions and contributed to the pathogenesis of pulmonary fibrosis (Hancock et al, 1986; Jones and Warren, 1992; Doherty et al, 1992). Thus, inhibition of macrophage infiltration and T cell activation in accelerated anti-GBM disease is a primary mechanism of DSG-mediated suppression of pulmonary haemorrhage and granulomatous lesion formation in this model.

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*r S OS 0 55 g *3 i Au 00 0 a ,SO V S Ah 1045E S TNFa is a proinflammatory cytokine produced by activated macrophages which can induce synthesis of a variety of cytokines such as IL-1 and IL-6 and induce leucocyte adhesion moleculE expression by endothelial cells (Jaattela et al, 1991). Recent studies have shown that intrapulmonary TNFa production participates in the pathogenesis of acute IgG immune complex alveolitis and 25 gram-negative bacterial pneumonia through a mechanism involving neutrophil recruitmenrt (Warren et al, 1989 a; Ulich et al, 1991). Also, intrapulmonary TNFa production has been demonstrated in the development of lung fibrosis and alveolitis in autoimmune pulmonary disease in lupus-prone mice (Deguchi et al, 1991) and in the development bleomycin-induced pulmonary fibrosis (Piguet et al, 1989). Thus, suppression of TNFa production is likely IA- to be a key mechanism by which DSG treatment suppressed both haemorrhage and the chronic phase of pulmonary injury.

"n addition, the failure to inhibit TNFa production at day 1 may explain why DSG treatment did not suppress acute neutrophil-mediated pulmonary haemorrhage or acute neutrophil-mediated glomerular injury occurring at this time (Lan et al, 1991 a; Lan et al, submitted for publication). The lack of DSG suppression at day 1 may reflect the kinetics of DSG action since it takes approximately 3 days to see an improvement in DSG suppression of acute allograft rejection episodes (Valdivia et al, 1990).

A number of studies have demonstrated a role for r, production of ROI in the pathogenesis of acute and chronic pulmonary injury, including development of hypersensitivity granuloiai and sarcoidosis (Warren et al, 1989 b; Guice et al, 1989; Cassatella et al 1989; Kasama ot al, 1992). The reduction in the percentage of cells spontaneously producing ROI at day 14 may by one mechanism by which formation of granulomatous lesions were prevented o by DSG treatment. However, this remains to be established a ,r and much of the spontaneous ROI production may have been involved in the destruction of phagocytosed red blood cells.

25 In this experimental model, DSG treatment did not suppress all functions of immune cells within the lung.

S Although DSG inhibited infiltration, immune-activation and TNFa production by pulmonary mononuclear cells, red blood cell phagocytosis and production of ROI were only partially affected. Indeed, the ability of pulmonary mononuclear cells isolated from DSG-treated animals to produce TNFU, in response to LPS stimulation demonstrated that these cells were not irreversibly inhibited. This result suggests that DSG may not suppress macrophage responses to major Ccam negative bacterial infection, which could be a potential advantage of DSG compared to conventional steroid and cytotoxic drug treatment of Goodpasture's syndrome where sepsis is a major cause of morbidity (Holdswoith et al, 1985).

Pulmonary injury in Goodpasture's syndrome is dependent upon the deposition of antibodies on the ABM (Koffler et al, 1969; Queluz et al, 1990). Inhibition of rat IgG deposition on the ABM and the GBM suggests that inhibition of the secondary antibody response is one mechanism of DSG-mediated suppression of pulmonary injury.

This is consistent with studies in which DSG suppress progressive slenomegaly, production of anti-DNA antibodies, and glomerular IgG and C3 deposition in S. spontaneous autoimmune lupus nephritis in susceptible mice (Okubo et al, 1988; Ito et al, 1990).

DSG is capable of suppressing progressive pulmonary haemorrhage, aggressive granulomatous lesion 20 formation and fibrosis in experimental Goodpasture's syndrome. DSG acted by suppressing both the local cell-mediated immune response and the systemic humoral immune response. Production of TNFa by pulmonary .o mononuclear cells is likely to play an important role in 25 the pathogenesis of pulmonary injury in this model, and inhibition of TNFa production may be a key mechanism of DSG action.

Treatment of Glomerulonephritis and Monitoring the Response Results from these animal experiments indicate that DSG could provide an effective clinical treatment for glomerulonephritis. The results of Examples 12, 14, suggest that DSG may be working by suppressing macrophage zAL function and immunoglobulin production in vivo. The potential indicated by Example 14 for analysis of patient serum and urine cytokine levels as an index of DSG effectiveness and disease development is a most exciting means by which clinical measurement of disease progression and immunological suppression can be linked.

Both glomerular and interstitial leucocytic infiltration '4lch are prominent features in this model will be significantly reduced. In particular, the degree of interstitial infiltration and its activation status correlates best with renal damage, and suppression of this may be a primary mechanism of DSG action.

From Example 11, it is anticipated that products of activated leucocytes, such as cytokines (IL-1, TNFa, IL- 6, etc.), will be significantly reduced. This has already been observed for TNFa, and is expected to be seen for other molecules. As DSG can act systemically, suppression of cytokine production is also likely to be systemic.

Thus, cytokine production in kidney, lung, blood, spleen, and lymph nodes may be reduced. This will be analysed by 20 both cytokine bioactivity and by gene expression.

DSG treatment has prevented the onset of renal impairment in this model using a 21 day experiment. It is anticipated that DSG would continue to provide protection from renal impairment, and experiments using longer timecourses will be performed. It is also anticipated that DSG will be able to suppress disease once it has been initiated.

4 4 4 4.

4 4 4 *4 *4 4 4.

44 0 4 4 4 4 There are a variety of ways in which DSG can potentially be administered. These include: Intravenous infusion; either for 3 hr periods or constantly; As an intradermal bolus; In an oral form that is absorbed from the gut; In an aerosol form taken by inhalation through the nose or mouth; and Administration of DSG encapsulated in a simple lipid vesicle/liposome would allow uptake by endocytosis and circumvent gut absorption difficulties. A DSG-liposome could be administered as an aerosol/ orally/ or subcutaneously. In addition, if it is desired to target DSG to a specific cell type, then specific ligands can be introduced into a non-endocytosed liposome.

A constant infusion osmotic pump placed subcutaneously, intraperitonealy, or connected intravenously may be used. A constant administration of DSG is likely to be more effective, and is likely to be of particular importance when treating disease already established.

5 Se a SS S 4* S. S S* S S S

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a s at 0 00 a 30 Without wishing to be bound by any proposed mechanism for the observed beneficial effect, DSG is anticipated to act to suppress leucocytic infiltration into the kidney, suppress leucocyte activation within the kidney and systemically, suppress B cell production of Ig, and T cell and macrophage production of pro-inflammatory cytokines.

This would have the effect of halting further renal injury, stabilising renal function, and possibly allowing a partial recovery of renal function as inflammation is suppressed and glomerular blood flow recovers. Haematuria may then resolve, and decreased Ig production may result in lower deposition of humoral immunoreactants in the glomeruli and interstitium, which would help to reduce the proinflammatory stimulus.

These effects could also be seen systemically, for example, a reduction of immune-complex deposition and decreased inflammation in other areas, such as joints and sites of tissue damage.

41 One interesting possibility is that DSG could act to halt the chronic sclerosis process that occurs within the kidney following the initial inflammatory event.

Progressive glomerular and interstitial sclerosis and glomerular crescent formation may be regulated by leucocyte populations. The effect of DSG on fibroblasts is unknown, but since mesangial cell proliferation can be partially inhibited by DSG, it is not unreasonable that fibroblast functions could also be affected. Thus, if DSG could limit the potentially damaging progressive sclerosis, it would be extremely useful in treating not just chronic nephritides, but may also be of use in a range of other autoimmune diseases where progressive tissue ablation by sclerosis occurs.

It will be clearly understood that the invention 0 in its general aspects is not limited to the specific 0 t details referred to hereinabove.

References cited herein are listed on the ofollowing pages.

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REFERENCES

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4. Cassatella G. Berton, C. Agostini, R. Zambello, Trentin, A. Cipriani "Generation of superoxide anion by alveolar macrophages in sarcoidosis: evidence for the activation of the oxygen metabolism in patients with high-intensity alveolitis" i.* Immunology, 1989 66 451-458 a 5. Dallman, M.L. Thomas, J.R. Green "MRC OX-19: A monoclonal antibody that labels rat T o lymphocytes and augments in vitro proliferative responses" Eur. J. Immunol., 1984 14 260-267 6. Deguchi S. Kishimoto d "Tumour necrosis factor/cachectin plays a key role in autoimmune pulmonary inflammation in lupus-prone mice" Clin. Exp. Immunol., 1991 85 392-395 7. Dijstra, e.A. dopp, P. Joling, G. Draal "The heterogeneity of mononuclear phagocytes in lymphoid organs: Distrinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2, ED3" Immunology, 1985 54 589-599 8. Doherty, N. Hirose, L. Zagarella, R.M. Cherriack "Prolonged monocyte accumulation in the lung during bleomycin-induced pulmonary fibrosis" Lab. Invest., 1992 66 231-242 9. Fujii, H. T. Takada, K. Nemoto, T. Yamashita, F. Abe, A. Fujii et a2 "Deoxyspergualin directly suppresses antibody formation in vivo and vitro" J. Antibiotics, 1990 43 213-219 10. Fukimoto, W.R. McMaster, A.F. Williams "Mouse monoclonal antibodies against rat major histocompatiblity antigen. Two Ia antigens and expression of Ia and class I antigens in rat thymus" Eur. J. Immunol., 1982 12 237-243 S 11. Guice, K.T. Oldham, M.G. Caty, K.T. Johnson, P.A. Ward "Neutrophil-dependent, oxygen-radical mediated lung injury associated with acute pancreatitis" Ann. Surg., 1989 210 740-747 44 12. Hancock, L. Kobzik, A.J. Colby, C.J. O'Hara, A.G. Copper, J.J. Godleski "Detection of lymphokines and lymphokine receptors in pulmonary sarcoidosis. Immunologic evidence that inflammatory macrophages express IL-2 receptors" Am. J. Pathol., 1986 123 1-8 13. Holdsworth, N. Boyce, N.M. Thomson, R.C. Atkins "The clinical spectrum of acute glomerulonephritis and lung haemorrhage (Goodpasteur's syndrome)" Q.J. Med., 1985 216 75-86 14. Holdsworth, N.M. Thompson, E.F. Glasgow, J.P. Dowling and R.C. Atkins.

"Tissue culture of isolated glomeruli in experimental crescentic glomerulonephritis" J. Exp. Med., 1978 147 98-109 Ishizuka, 14.

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Claims (13)

1. A method of treatment of an immunologically- mediated nephritis and/or an immunologically-mediLted lung haemorrhage comprising the step of administering to a mammal in need of such treatment an effective amount of a spergualin compound or of a derivative or salt thereof as the active agent.

2. A method of treatment according to claim 1 wherein the spergualin derivative is deoxyspergualin (DSG).

3. A method of treatment according to claim 1 or claim 2 wherein the nephritis is glomerulonephritis.

4. A method of treatment according to claim 3 wherein the glomerulonephritis is Goodpasture's syndrome. n 5. A method of treatment according to claim 1 a S wherein the immunologically-mediated lung haemorrhage is S" associated with c1o pasture's syndrome.

6. A method of treatment according to any one of claims 1 to 4 wherein the dosage range of the active agent US a a, is between 2.5 to 10 mg/kg of body weight.

7. A method of treatment according to any one of claims 1 to 6 wherein the active agent is administered in g combination with one or more other agents selected from dn iimmunosuppressive agents, lymphokines, cytokines or rn i differentiation factors.

8. A method of treatment according to claim 7 wherein the immunosuppressive agent is selected from one or more of prednisolone, cyclophosphamine, cyclosporine and

9. A method of treatment according to claim 7 wherein the cytokine-differentiation factor is selected from n GCr\-C.SFor G-CSF.

10. A method of treatment according to claim 7 wherein the differentiation factor is erythropoietin.

11. A method of treatment according to any one of claims 1 to 5 and 7 to 10 wherein tha dosage range of the active agent is up to at least 100 mg/kg of body weight. aA met' )d of monitoring the response -t erapy of an immunologically-mediated nephr it-ror immunologically- mediated lung haemorJ e disease which comprises measuring the lev cytokine in a biological fluid. ZL. A method of monitoring the response e:ahprny of e.pn-\hik-s or- nc\o\\k m.A\okd an immunologically-mediatea lung haemorrhage \sa oa method of treatment according to any one of claims 1 to 11 which comprises measuring the level of cytokine in a biological fluid. 4n-. 3 A method of monitoring the response to therapy according to claim 12 e-3e-6-wherein the biological fluid is selected from blood, plasma, serum, or urine. A method of monitoring the response to therapy a* according to claim 12 eF wherein the cytokine is selected from TNF-c( IL-1 or IL-6.

16. A spergualin compound or a derivative or salt 66 thereof when used in the method of treatment accor ng to any one of claims 1 to 11.

17. A spergualin compound accordin o claim 14 s wherein the spergualin derivative i (DSG).

18. A pharmaceutical omposition comprising as the active agent a spergu in compound or a derivative thereof according to clai 1 or 17 togrcher with a pharmaceutic Ly acceptable carrier and/or diluent, when used in e )ethod of treatment according to any one of cli, s 1 to 11. ABSTRACT A method of treatment of an immunologically- mediated nephritis and/or an immunologically-mediated lung haemorrhage comprises the step of administering to a mammal in need of such treatment an effective amount of a spergualin compound or of a derivative or salt thereof as the active agent. so A