Michael W. Whitehouse

Effect of adjuvant disease in rats on cyclophosphamide and isophosphamide metabolism

Abstract After the injection of a variety of arthritogenic adjuvants into male Wistar rats, hepatic activation of cyclophosphamide and isophosphamide is rapidly and profoundly depressed. This selective injury is largely reversible with phenobarbital and principally restricted to the liver microsomal protein fraction, which demethylates aminopyrine and N , N -dimethylaniline and generates “alkylating metabolites” from cyclophosphamide in vitro . Evidence is presented, based upon both metabolite excretion studies and the duration of hexabarbital-induced hypnosis, that this phenomenon is not an artifact in vitro and must be seriously considered in evaluating both the efficacy of potential anti-arthritic drugs against the rat adjuvant arthritis and their toxicity in these arthritic animals. A quantitative separation of two pathological responses to the same adjuvant may be obtained: (1) in Buffalo rats, whose liver metabolism may be profoundly impaired while they suffer minimal (or no) arthritis after being inoculated with adjuvants which are truly arthritogenic in other rat strains; (2) with Mycobacterium tuberculosis dispersed in methyl oleate, which induces minimal arthritis in Wistar rats, but nevertheless impairs their liver metabolism over a prolonged period (14 days or more). Drug metabolism appeared to be normal in rats with two other immunologically mediated “inflammatory diseases” (graft vs host disease and allergic encephalomyelitis) and in other rodents examined after adjuvant inoculations. A novel bioassay for cyclophosphamide and isophosphamide metabolites is described which utilizes their ability to prevent grafted rat lymphocytes from initiating a graft vs host reaction in tolerant recipient rats. At least four alkylating metabolites of cyclophosphamide were found in rat bile and tentatively identified by thin-layer chromatography. The possible error in relying on changes in urinary excretion (rather than biliary excretion) of drug metabolites as a guide to changes in hepatic xenobiotic metabolism is discussed.

Effect of cyclophosphamide on a local graft-versus-host reaction in the rat: Influence of sex, disease and different dosage regimens

A new pharmacological method for screening potential immunosuppressive drugs, the local graft-versus-host reaction in rats, has been used to evaluate the efficacy of cyclophosphamide applied at varous times in the development of this reaction.This drug was relatively ineffective when applied to the F1 hybrid recipient of the graft cells (splenic lymphocytes) prior to grafting, rather more effective when given only to the parental donor prior to harvesting the graft cells, and most effective when given to the recipient either immediately after the graft or after a delay of three days.Biliary and hepatic metabolites of cyclophosphamide diminished the competence of parental lymphocytes to evoke the graft response. By contrast, cyclophosphamide itself was devoid of such activity in vitro. Non-enzymic decomposition (hydroxylation) of cyclophosphamide with the Udenfriend system (Fe++, ascorbate, EDTA) efficiently generated in vitro graft-deactivating agents.Fortified liver preparations from normal female rats formed alkylating metabolites at a much slower rate, and adjuvant-arthritic male rats were less capable of generating graft deactivating cyclophosphamide metabolites in vitro, than liver preparations from normal male rats. However cyclophosphamide appeared to be no less effective in normal female or arthritic male rats in vivo, than in normal male rats, in controlling the graft-versus-host response. This lack of correlation between rates of hepatic cyclophosphamide metabolism and evident bioefficacy as an immunosuppressive drug is discussed, with special reference to similar findings by Sladek concerning rates of bioactivation and anti-tumor efficacy.

A case report: Thoracic duct lymphocyte drainage in rheumatoid arthritis

Abstract A 24-year-old woman with severe rheumatoid arthritis was treated by removing 170 × 109 lymphocytes through a thoracic duct fistula which drained continuously for 46 days. Marked clinical improvement occurred during the first 2 weeks of drainage and persisted until about 6 weeks after the fistula was discontinued, when articular inflammation recurred. Serum immunoglobulin concentrations and cell-mediated, transplantation, and humoral immune responses were suppressed in varying degrees after 4–6 weeks of drainage, but serum complement, inflammatory responses, and bone marrow granulocyte reserves were not adversely affected. Although the mechanisms responsible for the clinical response are not known; it is possible that cells contained in the thoracic duct lymph may have been continuously required in order to sustain the synovial inflammation of this patient.

Impaired Drug Metabolism in Rats Associated with Acute Inflammation: A Possible Assay for Anti-injury Agents

Summary 1. Many acute irritants, e.g., turpentine, mimicked Freunds adjuvant (FA) in causing rapid depression of hexobarbital and cyclophosphamide metabolism in rats in vivo. 2. Metabolism of cyclophosphamide and aminopyrine in vitro by liver homogenates was often subnormal if liver donor animals had been acutely inflamed (rear paw, tail) 48 hr previously. 3. Three classes of irritants/inflammagenic stimuli were distinguished by their effects on liver drug metabolism. 4. Carrageenan as routinely used for anti-inflammatory screening had a peculiar, selective effect on drug metabolism. 5. Drug metabolism was distinctly subnormal in animals inoculated with FA (rear paw, tail) at all stages prior to developing arthritis. 6. Drug metabolism was almost normal in prearthritic animals when FA was inoculated ip in a front paw or ear. Ear inoculations induced arthritis consistently in Lewis and Wistar rats. 7. Several drugs which prevented development of arthritis failed to normalize drug metabolism in the 4-day period after inoculating FA in rear paw or tail. 8. 6-MP and dexamethasone prevented the acute depression of drug metabolism and may be prototypes of a new class of anti-injury agents. We are grateful to Dr. C. M. Pearson for continued encouragement, the USPHS (NIH Grant GM 15759) for financial support and to Mrs. D. J. Whitehouse for surgical assistance.

Biochemical properties of anti-inflammatory drugs—XII: Inhibition of urate binding to human albumin by salicylate and phenylbutazone analogues and some novel anti-inflammatory drugs

Abstract We have previously reported that 0.2 mM phenylbutazone and salicylate inhibit the binding of urate in vitro to human albumin by over 50 per cent. This communication describes similar studies with over 60 other compounds. Only gentisate, per-fluorosalicylate, diiodosalicylate, salicylimide, Trimethazone, Diflumidone, the fenamic acids, 3-hydroxycinchophen and several acidic sulfonamides were as potent as phenyl-butazone or salicylate. A structure-action relationship was delineated for salicylate analogues. There was a consistent relationship between the ability of an acidic compound to displace albumin-bound urate and its relative avidity for the primary dansylamide (DNSA)-binding site(s) of the albumin. Some advantages of using DNSA, vis a vis other dyestufis, for measuring drug binding to albumin are discussed. Since some of the uricosuric action of certain drugs may depend on the displacement of urate from albumin or other proteins by the drugs, the assays in vitro for urate displacement which are described may facilitate pharmacological screening for potential uricosuric activity.

Interactions between non-steroidal anti-inflammatory drugs and biological membranes—II: Swelling and membrane permeability changes induced in some immunocompetent cells by various non-steroidal anti-inflammatory drugs

Abstract Non-steroidal anti-inflammatory drugs (NSAID) that are known to induce pseudo-energized high-amplitude swelling of mitochondrial preparations also induce swelling in various types of lymphocytes, namely: rat and rabbit thymocytes, rat and rabbit lymph node cells, and human and rat circulating lymphocytes obtained by thoracic duct drainage. The swelling of these immunocompetent cells was measured by light scattering. With the important exception of gold salts (no effect) and salicylates (very poor activity), almost all the NSAID that we have studied were able to induce cell swelling in vitro , but only at relatively high concentrations of these drugs compared to the concentrations which induced mitochondrial swelling (liver, kidney). The most dramatic effect was observed with N -arylanthranilates (fenamates), especially with flufenamic acid, which was used to study the mechanism of this drug action. The magnitude of the swelling is poorly related to the concentration of the drug, but the rapidity of this phenomenon seems to be a concentration-related phenomenon. If p -chloromer-curibenzoate ( p -CMB) or Mersalyl is present in the medium, the rate of NSAID-induced swelling is enhanced and appreciable cell swelling can even be demonstrated for drugs which otherwise (i.e. without p -CMB or Mersalyl) cause little cell swelling, e.g. phenylbutazone. By contrast, iodacetamide and N -ethylmaleimide fail to enhance the swelling. These activities seem to be related both to reactions with membrane thiol groups and modifications of the membrane charges by these compounds. The ionic composition of the medium is critical for this swelling activity and a cationic as well as an anionic selectivity was found for this phenomenon. These results suggest that one possible effect of the NSAID on leukocytes implicated in the immune and inflammatory responses may be on the permeability of the cell membrane to various ions.

Abnormal drug metabolism in rats after an inflammatory insult.

The drug-metabolising system (DMS) in rat liver microsomes is rapidly depressed after inoculating an inflammagenic adjuvant (e.g. Mycobacteria in oil). This is readily recognized by the prolonged sleeping time of adjuvant-treated rats injected with hexobarbital.In animals which develop adjuvant arthritis, the DMS is depressed for periods up to 6 weeks but returns to normal as the inflammatory phase of this chronic experimental arthritis is succeeded by the much less inflammatory osteogenic phase. Even mild, acute, irritants (such as turpentine or carrageenan) will induce changes in DMS within 24 hours.These findings indicate that ‘model’ diseases involving inflammation may affect the biodisposition of a drug in rats, often potentiating the drugs toxicity. The DMS in other rodent species appears to be very much less affected by an experimental inflammation. The effect of the ‘disease’ upon drug action, i.e. Pathopharmacodynamics, should be considered in evaluating new potential anti-inflammatory drugs, especially in rats. These changes in the DMS may perhaps be caused by the same mediators (toxohormone?) which rapidly stimulate hepatic fibrinogen synthesis, in response to many forms of inflammation.

The local graft versus host reaction in the rat as a tool for drug mechanism studies

1 A local graft versus host reaction in rats permits the evaluation of drug action on (i) donor (parental) animals and (ii) recipient animals (F1 hybrid) prior to establishing a lymphoid cell graft to detect drugs which might attenuate immunocompetence over a prolonged period (e.g. steroids). 2 Treatment of the recipient animals after a lymphoid graft readily detects anti‐proliferative agents and may disclose other agents which interfere with the recognition of non‐self by viable graft cells. 3 Treatment of the cell graft in vitro before innoculation into the recipient animal permits the detection of alkylating agents, wihch effectively deactivate graft cells at non‐toxic concentrations. 4 Examples are given of how this reaction may be used to evaluate functionally the effects of chemical modification of the cell surfaces of lymphocytes and to detect active metabolites generated from a precursor drug (e.g. cyclophosphamide). 5 Some novel immunosuppressant drugs are described.

I. Drug Sensitivity of Rat Adjuvant Arthritis, Induced with ‘Adjuvants’ Containing no Mineral Oil Components

Summary 1. Adjuvant arthritis induced with mineral oil adjuvants was resistant to acetylsalicylic acid and flufenamic acid when given prophylactically, and only moderately sensitive to phenylbutazone. 2. Adjuvant arthritis induced in the same rat strain with 4 metabolizable adjuvants (M. tuberculosis with squalene or squalane or butyl palmi-tate or butyl stearate) was considerably more sensitive to acetylsalicylic acid, phenylbutazone and flufenamic acid. 3. These alternative adjuvants offer promise for screening weaker, aspirin-like (antiarthritic) drugs in rats, to derive less toxic (ulcero-genic, leukopenic) drugs for use in man. We are grateful to Dr. C. M. Pearson for continued encouragement, the USPHS (NIH grant GM 15759) for financial support, to Drs. R. Per-rin and J. K. Pollard, (Calbiochem) for gifts of butyl palmitate and commercial adjuvants (Per-rigens), and Mr. J. Fitzgerald, Miss R. Stremel and Mrs. E. Taylor for their unfailing technical assistance.

Some (pharmacological) properties of chloracetaldehyde, an oxidation product and potential metabolite of cyclophosphamide

Circumstantial evidence is presented that chloracetaldehyde is generated from cyclophosphamide by chemical oxidation in vitro with peroxide and might possibly be formed in vivo as a product of cyclophosphamide bioactivation.Chloracetaldehyde has several attributes of an agent capable of suppressing cellular immunity. Thus it prevents a graft-versus-host reaction (both in vivo and in vitro), is cytostatic and under certain conditions can abrogate an ongoing immunopathy (EAE).These properties are also exhibited by HN-2 and certain alkylating metabolites of cyclophosphamide.