The First Step: Defining Disease Characteristics

Definitive histomorphologic characterization of hepatobiliary disease (routine and special stains for fibrosis, metals, infectious agents), combined with tissue culture (aerobic and anaerobic bacterial), cytologic imprints of biopsy specimens (may disclose infectious agents not recognized on histology), and quantitative metal analyses (copper [Cu], iron [Fe], zinc [Zn]) are essential for selection of interventional therapies. It is important to recognize that there are no controlled trials or even long retrospective studies on response to specific interventional approaches to most disorders in dogs and cats.

The Second Step: Consider Pathomechanisms of Liver Injury

Oxidative Injury: This is complex involving cellular and molecular mechanisms that initiate and perpetuate damage that leads to liver injury and fibrosis. Oxidative injury and production of reactive oxygen species [ROS] are central pathomechanisms in most forms of acquired liver injury.

Toxins, Endotoxins, Infectious Agents: The central role of the liver in intermediary metabolism and detoxification processes, its large resident macrophage population (Kupffer cells = 80% of the fixed macrophages in the body) and its sentinel position between the splanchnic and systemic circulatory systems puts the liver at high risk for toxic, infectious, endotoxin, and oxidant mediated injuries. Receiving 75% of its blood flow directly from the alimentary canal (the richest source of oxidants, invading bacteria, and toxins), both acute and chronic enteritis are thought to substantially contribute to liver injury. Pancreatic inflammation (involving parenchyma or ducts) imposes risk for obstructive cholestasis and hepatobiliary inflammation. A variety of toxins have been identified that specifically impose liver injury, including certain drugs: NSAIDs, acetaminophen, phenobarbital, primidone, diazepam (cat), bacteria, enterotoxins, endotoxins (LPS), toxins derived from molds, fungi, algae, spoiled or contaminated food, or effects of consumed transition metals.

Cholestatic Liver Disease: A heterogeneous group of disorders associated with impaired bile flow. In severe hepatic insufficiency (associated with acquired portosystemic shunting [PSS] secondary to intrahepatic fibrosis and portal hypertension), and in patients with other forms of cholestasis, accumulation of membranocytolytic bile acids (BA) (hydrophobic BA) contributes to ongoing hepatocellular injury. Noxious BA damage cell and organelle membranes, induce intracellular structural and functional change, inflammation, and compromise bile flow. Free radicals also contribute to cholestatic injury; a central mechanism of BA hepatotoxicity is reduction of mitochondrial glutathione (GSH) resulting in reduced production of cell energy. Polymorphonuclear leukocytes (neutrophils) contribute to tissue injury in many cholestatic conditions, also contributing to oxidant injury. Likewise, hepatocellular copper (Cu) retention (due to impaired Cu egress in bile) and iron (Fe) accumulation (in macrophages secondary to inflammation) also contribute to ROS generation. Sulfation of membranocytolytic BA reduces their toxicity and facilitates BA renal elimination. This occurs extensively cats but not dogs. Taurine conjugation enhances physiologic digestive functions of BA and slows their passive intestinal absorption (small bowel). Taurine is thought to attenuate (to some degree) hepatocellular BA toxicity, for a variety of reasons and may be helpful when ursodeoxycholic acid is chronically administered to patients with impaired bile acid elimination.

Immune- Mediated Mechanisms: Are thought to perpetuate chronic necroinflammatory / cholestatic liver injury and to augment injury instigated by infection, endotoxins, or obstructed bile flow. A variety of pathologic immunologic responses perpetuate inflammation that ultimately impose oxidative insult. Phenomena thought to unite infection and "auto"immune responses include molecular mimicry (antigens of the infectious agent closely mimicking self-antigens) or innocent bystander effects (exposure or mobilized self-antigens). These responses may culminate in a learned immune repertoire involving T-cells and B-cells ultimately targeting foci on normal cells. Infectious agents may initiate or aggravate responses through an adjuvant effect, providing co-stimulatory inflammatory signals or by functioning as superantigens capable of broadly activating T cells. Environmental factors and toxins also have been implicated in induction of chronic immune responses in humans that demonstrate "auto"immune behavior.

Transition Metals: Copper (Cu) & Iron (Fe): While these metals function as important catalysts for enzymes and reactions essential to health, pathologic hepatic accumulation imposes oxidant injury. Mitochondria are a primary site of injury where impaired GSH concentrations disrupt cell energy production.

Hepatic Iron: Fe accumulates in macrophages in many necroinflammatory disorders (80% biopsies). While older theories proposed that such sequestered Fe is unable to catalyze free radical reactions, recent data refutes this dogma. Fe is constantly in flux within and between cells and even the minute hepatocellular pool of free Fe participates in free radical reactions. Excess Fe also initiates/ promotes fibrogenesis. In the presence of toxins, Fe hepatotoxicity is enhanced (e.g., endotoxin imposes a "priming" effect on Kupffer cells).

Hepatic Copper (Cu): While increased liver Cu concentrations may derive from genetic (transport/storage) disorders, it is more common secondary to cholestasis in dogs. Normally, Cu absorbed from the gut circulates bound to transport proteins. After hepatic uptake and binding to cytosolic proteins, Cu is used in enzyme pathways and ultimately stored affiliated with metallothionein an important thiol bearing protein. Normally, Cu excretion into canalicular bile and enterohepatic circulation of Cu regulates a neutral Cu balance. However, cholestasis from any cause impedes biliary Cu excretion causing eventual lysosomal loading. This leads to organelle damage secondary to cell membrane oxidation. Rupture of lysosomes leads to hepatocyte death. Accumulation of Cu storage "granules" (Cu-binding protein) can be identified on routine H&E histology. Such identification is commonly "over-interpreted" as Cu storage disease (genetic) prompting recommendation of chelation therapy.

How to Define Transition Metal Associated Liver Injury? Special stains (Prussian blue for Fe; Rhodanine or Rubeanic acid for Cu) must be reconciled with quantitative metal analysis (ug/gm dry weight tissue) and histologic interpretation of a biopsy. Histology defines the acinar distribution of Cu or Fe (macrophage, hepatocyte). Very small liver biopsies can yield erroneous measured metal values and histologic interpretations; e.g., biopsy of only a regenerative nodule may misrepresent tissue activity engaged in the disease process (regenerated tissue has low metal relative to actively diseased tissue). Quantitative tissue Cu measurements reconciled with histology define the need for chelation therapy or Zn supplementation and antioxidants (high Cu or Fe liver concentrations indicate a need for Vit. E and a thiol donor).

The Third Step: Categorizing Disorders (common): Pathomechanisms Linked to Interventional Rx

Necroinflammatory: Chronic hepatitis (CH, canine); cholangiohepatitis (CCHS, cat); extrahepatic bile duct occlusion (EHBDO); lobular dissecting hepatitis; repeated toxin induced injury. Rx: immunomodulation, antioxidants, UDCA, ±antifibrotics (based on histology).

Cholestatic: Parenchymal disorders with hyperbilirubinemia or high BA, bile duct focused disorders: CCHS, cholangitis, EHBDO, biliary mucocele, hepatocellular dysfunction / canalicular collapse: hepatic lipidosis (HL, cats), severe vacuolar hepatopathy (VH, dogs). Rx: correct mechanical obstruction, rectify mucocele, Rx: UDCA, SAMe (antioxidant, other effects), antioxidants, + taurine (esp. cat).

Metal Associated: Inflammatory / cholestatic disease with high Cu or Fe concentrations, +/- Zn depletion. Rx: If high Cu (> 1,500 ug/gm dry wt liver): chelation, restrict Cu intake in food & water, anti-oxidants, Zn supplementation not concurrent with chelation; if Zn depletion (< 120 ug/gm dry liver): supplemental Zn (common if PSS, feeding protein restricted diets).

Fibrogenesis: Non-inflammatory: juvenile fibrosing hepatitis; inflammatory: CH, chronic CCHS, chronic EHBDO. Rx: Polymodal therapy recommended: immunomodulation, antioxidant, Vitamin E, Silibinin, polyunsaturated phosphatidylcholine or colchicine. Colchicine inhibits neutrophil function & collagen deposition and can impose important side effects if dosed too high.

Hepatotoxicity: Multiple toxins / drug toxicities described. Rule out: infection (serum titers tissue cultures), Environmental toxins: food, garbage, contaminated water (e.g., cyanobacteria/algae). Rx: Suspend toxin exposure, appropriate enteric removal (emesis, colonic lavage, activated charcoal avoiding repeated dosing with sorbital), discontinue suspected toxin, research toxic mechanisms (Internet, PubMed), give appropriate antidote(s). Do not give prednisone, rather remove toxin and facilitate toxin excretion / removal (e.g., acetaminophen: administer cimetidine to slow biotransformation to toxic adduct. Generally, use hepatoprotectants and antioxidants: n-acetylcysteine (NAC), SAMe, silibinin, and Vit. E. For mushroom toxicity (amanita, death cap) give silibinin, penicillin (impairs cell toxin uptake), and antioxidants.

Portosystemic Vascular Anomaly (PSVA): Congenital macroscopic portal shunting "around" the liver; congenital microvascular intrahepatic shunting (microvascular dysplasia, MVD). No necroinflammatory or cholestatic pathomechanisms involved. Rx: PSVA: surgical ligation or medical management of hepatic encephalopathy, Zn supplementation. MVD: usually requires no therapy. UDCA NOT indicated in most patients, antioxidant not indicated in most patients.

Biliary Mucocele: Inspissated biliary material (gallbladder [GB], "kiwi" fruit pattern on ultrasound, associated with GB cystic mucosal hyperplasia, GB hypokinesis, hyperlipidemia, vacuolar hepatopathy, and sometimes cholecystitis and ruptured GB; may also affect common ducts and hepatic ducts. Rx: Remove inspissated biliary material, GB resection usually indicated, induce hydrocholeresis: maintain good hydration, UDCA, and SAMe. Culture bile/tissue: aerobic and anaerobic bacteria, inspect cytology of bile for bacteria (may see bacteria that fail to grow in culture due to antibiotic treatment preceding sample acquisition). UDCA promotes bile flow and aids elimination of other substances excreted in bile (up-regulation of canalicular transport and stimulated ductal bicarbonate secretion). SAMe may augment bile flow via enhance GSH biliary concentrations (GSH fuels non-BA dependent bile flow). UDCA: 15 mg/kg PO SID, SAMe: 20 mg/kg PO enteric coated tablets on empty stomach. Appropriate antibiotics, fat restricted diet if hyperlipidemia associated with VH. Vitamin E if inflammatory histology.

Non-Necroinflammatory Disorders Secondary to Systemic or Metabolic Disorders

Vacuolar Hepatopathy (VH): In dogs, hepatocyte distention with cytosolic glycogen secondary to chronic stress imposed by systemic or hepatic disease. Relates to chronic release of inflammatory cytokines (dental disease, IBD, skin infections, neoplasia), or Hyperadrenocorticism (iatrogenic, spontaneous typical hyperadrenocorticism, or sex hormone adrenal hyperplasia). Rx: Identify and treat underlying disease process; e.g., sex hormone related adrenal hyperplasia treated with lysodren rather than trilostane. If VH diffuse and associated with stromal collapse, high serum or urine bile acid concentrations.

Hepatic Lipidosis (HL) Cats: Triglyceride distention of hepatocytes, secondary to anorexia and another primary disorder. Rx: identify underlying cause of anorexia, provide protein replete feline diet, NAC (140 mg IV over 20 min., then 70 mg/kg IV q6-12 hrs, correct hypokalemia and hypophosphatemia, beware of electrolyte changes with re-feeding phenomenon, supplement with taurine (250 mg PO SID to BID), l-carnitine (250 mg PO SID [use Carnitor®), vitamin E (10 IU/kg/day), water soluble vitamins, determine B12 status, treat while awaiting data (1.0 mg/cat, SC).

"Reactive" Hepatitis: Diagnosed subsequent to liver biopsy for unexplained liver enzyme activity; a term applied to liver biopsies lacking a distinct pattern but showing multifocal lipogranulomas (small clusters of macrophages with Fe [hemosiderin], minor lymphoplasmacytic portal infiltrate, but lacking overt necrosis, fibrosis, or architectural remodeling. Rx: Reactive hepatitis is not a disease but merely represents the sentinel function of the liver. Beware of recommendations to intervene with anti-inflammatory / immunomodulatory treatments if morphologic description seems vague. Call and talk with your pathologist before committing to the chronic use of potentially toxic drugs.

Specific Considerations Interventional Strategies

Nutrition

Balanced nutritional support is critical including vitamin supplements (avoid Cu supplement if high tissue Cu). Only restrict protein in patients showing signs of HE (may be vague, may be indicated by ammonium biurate crystalluria, cannot depend on blood ammonia determinations). Most animals with acquired hepatobiliary disease do not require protein restriction, especially cats. Cats with HL may succumb subsequent to dietary protein restriction.

Antioxidants

Approximately 65% of dogs and cats with necroinflammatory liver disorders have low liver GSH concentrations.. Since oxidant injury is better inhibited than reversed, early preemptive therapy in necroinflammatory and cholestatic liver disease may be most effective. Antioxidant therapy should be combined with disease appropriate immunomodulatory / anti-inflammatory / antifibrotic medications to achieve a synergistic effect. For example, glucocorticoids intervene in membrane release of arachidonic acid that initiates production of inflammatory eicosanoids that play a crucial role in membrane oxidation. Thus, concurrent of an antioxidants may yield a synergistic benefit.

Direct Thiol / Glutathione Donors--N-Acetylcysteine, S-Adenosylmethionine, Whey Protein ?, Silibinin (Milk Thistle), N-Acetylcysteine (NAC): Used IV for crisis intervention, especially during the first few days in cats with HL, and in animals with suspected of hepatotoxicity. Dose: 140 mg/kg IV (dilute at least 1:4 with saline or 5% dextrose, give via 0.25 μ micron nonpyrogenic filter), administer over 20-min NOT as constant rate infusion (CRI). Follow-up dosing: 70 mg/kg IV given 2-4 times daily as clinically indicated.

S-Adenosylmethionine (SAMe): For necroinflammatory / cholestatic liver disease / VH / HL: Broad metabolic benefits may have important metabolic implications as a GSH donor, for methylation reactions (including l-carnitine and phosphatidylcholine synthesis). In HL, low vitamin B₁₂ may contribute to SAMe and GSH deficiency (compromised methionine availability for transsulfuration pathway). Dose: 20 mg/kg enteric coated tablets, given on an empty stomach. Be particular about source, Denosyl-SD4^TM, Nutramax, Inc has proven bioavailability and increases hepatic GSH in healthy cats, cats with portal triad inflammation, and dogs treated with high dose glucocorticoids.

Whey Protein: Alternative cysteine source for GSH synthesis; other nutritional benefits claimed but efficacy remains unproven. Consult recent review: Marshall K: Therapeutic applications of whey protein. Altern Med Rev. 2004;9:136-156. Product labeled Protectamin (Fresenius Kabi, Bad Hamburg, Germany) may provide greater GSH substrates.

Vitamin E (α--tocopherol): "last line of membrane defense" as lipid peroxidation chain terminator. In membranes exists in low molar ratio: phospholipids (especially PUFA) that are highly susceptible to oxidation (1,000-2,000 PUFA:1 Vit. E). Efficient recycling of oxidized (tocopheroxyl radical) to reduced antioxidant form depends on an interactive group of redox antioxidant cycles (CoQ₁₀, ascorbate, GSH). Large doses without interactive antioxidants may fail to provide expected antioxidant protection and may become pro-oxidant (accumulated tocopheroxy radical). Vit. E also modulates cellular responses to oxidative stress through signal-transduction pathways (protein kinase C) providing anti-inflammatory and antifibrogenic effects (reduces collagen gene transcription). Since it is not synthesized in vivo it must be ingested (diet or supplementation). Dose: 10 IU/kg PO / day (α--tocopherol acetate); higher dosing for bile duct occlusion or cats with severe sclerosing cholangitis (fat malassimilation due to disrupted bile acid enterohepatic circulation). Water-soluble (α--tocopheryl succinate polyethylene glycol 1000 [TPGS]) form preferred if compromised fat uptake (TPGS forms micellar solutions at low concentrations obviating need of bile acids for Vit. E uptake). Toxic effects of Vit E if very large doses: may potentiate oxidant injury and interferes with Vit. K activity (bleeding tendencies).

Silibinin / Silymarin (Milk Thistle)

Studied in a considerable repertoire of clinically relevant live animal disease models. Proven for prevention / recovery from certain toxins (e.g., amanita mushroom, CCl₄, ethanol). Despite numerous studies in humans, clinical benefit in chronic liver disease remains controversial. Imparts antioxidant, anti-inflammatory, and anti-fibrotic effects, promotes protein synthesis (regeneration), and interferes with certain p450 enzymes and toxin uptake/ activation. Hepatoprotective, anti-inflammatory, antifibrotic, and antioxidant effects mechanistically overlap with several other nutraceuticals, (specifically Vit. E, SAMe, NAC, and polyunsaturated phosphatidylcholine). Avoid mixed herbal formulations of milk thistle, except combination with polyunsaturated phosphatidylcholine. Later combination has superior advantage in hepatic disease. Dose of silibinin complexed with PPC: 2-5mg/kg per day (based on experimental work in other species; current veterinary product provides Vit. E 300 IU. Zinc gluconate 45 mg with 70 mg silibinin complexed with phosphatidylcholine.

Ursodeoxycholic Acid (UDCA)

Least controversial mechanism involves protection against membranocytolytic bile acids in liver, bile, and systemic blood providing direct cytoprotection (hepatocellular membranes, possibly blood brain barrier) and molecular interventions accentuating survival signals. May suppress MHC expression (target foci, MHC II) on hepatobiliary surfaces. Also is immunomodulatory and produces hydrocholeresis that may aid in biliary toxin elimination. Recommended in chronic necroinflammatory and cholestatic liver disease, cholestatic disorders complicated by "sludged" or lithogenic bile, but no evidence of benefit in acute toxic injury or HL in cats. Biliary diversion/decompression necessary if bile duct occlusion before UDCA therapy. May blunt peribiliary inflammation and fibrosis in EHBDO. Unjustified for congenital portosystemic shunts, limited benefit (if any) in short term jaundice due to sepsis or cats with HL.

Cu Storage Rx

Reduce Cu intake (water, food), use chelation, and impair enteric Cu uptake (zinc) to reduce Cu accumulation. Chelation: indicated when tissue Cu > 1,500 ug/gm dry weight liver. Preferred chelator: d-penicillamine (15 mg/kg PO BID, 30-minutes before meals with supplemental pyridoxine). If patient intolerant, Trientine may be used (author has observed Trientine associated acute renal failure in 2 dogs). Chelation for at least 6-months; second liver biopsy used to determine best chronic treatment. If Cu is critically lower: may convert to chronic zinc acetate (gluconate or sulfate). If patient zinc intolerant, use chronic chelation (d-Pen. dose reduced by 50%). Do not use chelation and Zn together, direct chelation of zinc to d-Pen. Avoid ascorbate supplementation as this may enhance transition metal oxidative injury. All patients with high liver Cu should receive supplemental Vit. E as an antioxidant antifibrotic.

Zinc Supplementation

Essential trace element, required for many homeostatic functions with central importance to the liver, e.g., normal protein metabolism, function of >300 zinc metalloenzymes, and membrane integrity. Zn sufficiency has impact on numerous physiologic reactions including: immune and neurosensory functions, detoxification pathways, wound healing, appetite, imparts antioxidant effects reducing some but not all ROS mediated injury (antagonizing redox-active transition metals: Fe, Cu which it competitively displaces). Zinc insufficiency increases susceptibility to GSH deficiency. Liver Zn should be measured concurrent with both Cu and Fe in liver biopsies. Supplement Zn when concentrations < 120 ug/mg dry liver, especially when either Fe or Cu values are high. Low tissue Zn concentrations are common in animals with severe liver disease, especially with acquired/congenital PSS. Feeding a restricted protein diet may augment this phenomenon. Association between low tissue Zn and GSH in severe liver disease suggests greater risk for transition metal injury. Zn therapeutically impedes hepatic Cu accumulation (liver binding, enteric uptake). When used for transition metal injury, concurrent administration of Vitamin E and a thiol donor (SAMe) are recommended (synergistic effects). Dose: 1-3 mg/kg elemental zinc if low tissue zinc; 3-10 mg/kg elemental zinc for Cu toxicity (30-min. before feeding).

Immunomodulation

Azathioprine: Used for CH and lobular dissecting hepatitis when infectious cause unlikely or eliminated; reserved for dogs. Antimetabolite: impairs purine metabolism. Dose: 1-2 mg/kg PO SID for 3-4 days then every other day (EOD). Cats do not tolerate this drug. Toxicity: hematopoietic (acute or chronic bone marrow toxicity: leukopenia, thrombocytopenia) and gastrointestinal signs (vomiting and diarrhea); occasional side effects: pancreatitis, dermatologic reactions, and rare hepatotoxicity (cholestasis rare in dogs, veno-occlusive lesion in human beings). Acute bone marrow toxicity requires suspending treatment, await recovery, continuing treatment with 50% or 75% initial dose. Chronic bone marrow toxicity, pancreatitis, hepatotoxicity: permanent drug discontinuation, use alternative immunomodulatory agent (e.g., mycophenolate) Good clinical response allows 50% dose reduction. Patient response: monitored initially at weekly (4 weeks) then quarterly (CBC, liver enzymes, total bilirubin). Many patients requiring prednisone and azathioprine for control of chronic liver disease cannot be completely weaned off either drug. Chronic therapy is combined with antioxidants (Vit. E, SAMe), and UDCA (if high serum BA).

Mycophenolate Mofetil: Morpholinoethyl ester pro-drug of mycophenolic acid (MPA), a selective potent inhibitor of inosine monophosphate dehydrogenase (IMPDH). This enzyme is critical for de novo synthesis of guanosine triphosphate (GTP), a purine necessary for synthesis of DNA, RNA, proteins, and glycoproteins. MPA is relatively selective for lymphocytes which are dependent on a purine synthetic pathway inhibited by MPA permitting targeting of activated lymphocytes (inhibits clonal expansion: B and T lymphocytes, antibody production, and expression of lymphocyte cellular adhesion molecules). MPA is eliminated after hepatic glucuronidation (inactive MPA-glucuronide); dose in canine liver patients used by the author. Dose: 10-20 mg/kg PO BID (20 mg/kg q 12 hours proven successful for myasthenia gravis in dogs). Reduce dose for long term treatment (after remission) by 50% (5-10 mg/kg PO BID). Alternative for dogs intolerant of azathioprine (these may have impaired azathioprine metabolism, pancreatitis, hepatoxicity, bone marrow toxicity). Rare bone marrow suppression (humans, dog). Limited information for cats.

Metronidazole: Provides bactericidal, amebicidal, trichomonacidal, cytotoxic, immunosuppressant (cell mediated immune responses) influences and a dose dependent antioxidant effect. Used adjunctively with other drugs (e.g., prednisone) improves response compared to single agent therapy (man). Especially recommended for liver disease associated with inflammatory bowel disease. Limited protein binding allows delivery to bile, bone, effusions, CSF, and hepatic abscesses. Relies on hepatic metabolism (30-60%) along with renal and fecal elimination; dose adjustment required if compromised liver function. Dose: Empirical dose reduction in liver disease (used in veterinary patients with good success for > 17 years): 7.5 mg/kg PO BID. Side Effects: anorexia (metallic taste) and neurologic effects with excessive dosing; vestibular signs most common and usually resolve within 1 week of drug discontinuation or dose adjustment. Antagonize neurologic toxicity (rapid recovery) with diazepam unless HE.

Methotrexate: A folic acid antagonist, that reversibly and competitively inhibits dihydrofolate reductase; undergoes renal elimination. Used as a "rescue" or second line anti-inflammatory- immunomodulator in a variety of immune mediated diseases in humans. Active against cells in which it becomes trapped by polyglutamination; is concentrated in lymphocytes, perhaps in biliary epithelium (limited evidence), and achieves concentrations in bile. May offer "targeted" therapy in lymphocyte mediated chronic biliary inflammation. Useful in some humans with sclerosing cholangitis, but response is inconsistent. Proven useful in some cats with sclerosing cholangitis as these typically do not respond to prednisolone as single agent immunomodulatory, and appear predisposed to diabetes mellitus (with prednisolone therapy). Only use when biopsy proves diagnosis of sclerosing CCHs, infection ruled out, and with polymodal therapy: UDCA, SAMe, Vit. E, low dose prednisolone, and supplemental folate. Author prefers methotrexate to chlorambucil in these cats. Positive response indicated by declining bilirubin concentrations; low level enzyme activity usually continues. Dose: use only with low dose pulse therapy regimen (treat once weekly) to avoid side effects; 0.4 mg total dose divided into 3 doses given on one day at 8 hour intervals once weekly. Formulate capsules to 0.13 mg for once weekly single day pulse dosing. Renal disease reduces clearance and can result in drug accumulation. Use ONLY with folate supplementation (0.25 mg/kg folate or folinic acid daily). May be used IM or IV but reduce dose by 50% if administered using these routes. Very immunosuppressive and increases risk of infection (infections observed by author: feeding tubes, pyelonephritis, Demodex, herpes keratitis). For polymodal CCHS treatment in cats, methotrexate replaces azathioprine or chlorambucil. Estimated canine dose: 0.1 mg/kg over 24 hours (divided into 3 doses) repeated q 7 to 10 days?

Alternative immunomodulators not discussed here: chlorambucil, cyclosporine.

Antifibrotics

A number of agents provide antifibrotic influences: including antioxidants (SAMe, Vit. E), UDCA, and Silibinin. However, primary antifibrotics are: polyunsaturated phosphatidylcholines (PPC) and colchicine.

Polyunsaturated Phosphatidylcholines: Extract of soybeans or salmon roe; a mixture of seven phospholipid species rich in polyunsaturated phosphatidylcholines, sometimes classified with the group of B-vitamins. Active component: dilinoleoyl-phosphatidylcholine (DLPC) accounting for approximately 50% (w/w) of polyunsaturated phosphatidylcholine. Attenuates hepatic fibrosis in a number of animal models and in humans with chronic active hepatitis. Mechanisms: hepatoprotectant, anti-inflammatory, immunomodulatory (glucocorticoid sparing effect permitting dose reduction when adjunctively used with prednisone and azathioprine), and antioxidant effect. DLPC may directly influence hepatocyte cell and organelle membrane structure (membrane stabilizing effect), directly attenuate transformation /activation of Stellate cells into myofibrocytes (source of liver collagen), increases Stellate cell collagenase activity (digests collagen), and reduces platelet derived growth factor stimulation of Stellate cells; all these effects are proven to contribute to hepatic fibrogenesis. Study of DLPC and SAMe (ethanol hepatotoxicity) demonstrated similar lipid membrane enrichment, but DLPC minimally attenuated liver enzymes and cholestasis compared to SAMe. With an in vitro testing system, DLPC did not, but SAMe did restore total cell and mitochondrial GSH and improved organelle / cell oxygen consumption. SAMe provisioning the transmethylation pathway enables hepatocyte phosphatidylcholine synthesis and thus may provide similar effects. DLPC is derived from PPC after enteric digestion, PPC circulation to the liver, and reservoir-like incorporation in membranes and lipoproteins. Dose: 50 to 100 mg/kg PO SID (no greater than 3 gm suggested in man) extrapolated for dogs and cats by the author from human and experimental animal studies. No side effects have emerged from use > hundred dogs with liver disease and fewer cats.

Colchicine: Inhibits microtubular apparatus, may arrest hepatic fibrogenesis by interfering with transcellular movement of procollagen fibrils, inhibiting procollagen synthesis, and fibroblast proliferation, and by inducing collagenase. Imposes antiinflammatory effects by suppressing leukocyte locomotion and degranulation, and impaired expression of surface TNF receptors. May facilitate hepatic Cu excretion. Inconsistent benefits shown in humans with chronic liver disease. Used primarily in juvenile fibrosing liver disease in dogs where there is little inflammation. Toxicity: hemorrhagic gastroenteritis, bone marrow suppression, renal injury, and peripheral neuropathies. Considered a safer therapeutic alternative than D-Penicillamine or glucocorticoids in man. Colchicine commonly combined with probenecid which increases its duration of action. The form without probenecid is preferred in liver disease since biotransformation and elimination of colchicine is in part dependent on the liver. May undergo reservoir accumulation (proven in humans). Owners must be warned of potential teratogenic and abortifacient effects (contact through urine and tablet, provide written warning to pregnant women). Use concurrent with UDCA, glucocorticoids, and antioxidants. Not recommended as concurrent treatment with azathioprine, methotrexate, or chlorambucil owing to similar side effects; rather use PPC in these. Dose: 0.025-0.03 mg/kg PO SID (probenecid free drug); used in many dogs and fewer cats without problems.