8 Amphotericin B

JOS F. BERNARDO, RAMZI SABRA AND ROBERT A. BRANCH

1. Introduction
In recent years, systemic mycoses have become a prominent cause of disease particularly in severely and immunocompromised patients. The factors contributing to the increased prevalence of fungal infections are related to larger number of patients with underlying immunosuppression, due to factors such as the acquired immunodeficiency syndrome (AID !, more aggressive cancer chemotherapy, increase in transplantation operations, greater number of other immunocompromised patients, and new and more prosthetic devices "#$. There have been a number of recent surveys which give some illustration about this problem. The %enter for Disease %ontrol reported that among &# ' A hospitals, candidiasis was the eighth most common infection, accounting for &( of the isolates ")$. This value can be considerably higher in certain specific patient groups. The *ational %ancer Institute estimated that +,( of patients dying with acute leu-emia had systemic fungal infection at auto. psy ",$. In patients with AID , the most common fungal infection is oropharyngeal candidiasis. /ow. ever, in these patients, the fungal infection with the highest mortality rate is cryptococcosis. It can be clearly seen from these e0amples that the li-elihood of a systemic fungal infection is an important consideration in the treatment of a severely ill and immunosuppressed patients "+$.

Amphotericin 1 has remained the mainstay of therapy for serious fungal infections since its intro. duction in #2&3, despite the availability of newer agents "&$. The usefulness of this agent, however, is compromised by the frequent occurrence of several acute and chronic adverse effects that often necessitate changes in, or premature discontinuation of, therapy. These include fever, chills, nausea, vomiting, anore0ia, headache, bronchospasm, hypotension, anaphyla0is, and bone marrow suppression. The most limiting adverse effect, however, is nephroto0icity. Amphotericin 1 is a member of the polyene macrolide class of antibiotics. The molecule consists of a large macrolide lactone ring of ,4 carbon atoms, one side of which is composed of a rigid lipophilic chaira of seven con5ugated double bonds, and the opposite side of a similar number of hydro0ylated carbon atoms (6ig. #!. Thus, the molecule is amphipatic, and this feature of its structure is believed to be important in its mechanism of action "3$. The ma5or action of amphotericin 1 is believed to be on the cell membrane of fungal and mammalian cells. The drug binds to sterols in the cell membrane and induces formation of aqueous pores which result in impairment of barrier function and loss of protons and cat7ons from the cell. At low concentrations, the increased permeability is restricted to small molecules or cations such as sodium and potassium. At higher concentrations or after prolonged incubation, other cell constituents are lost

and this leads to metabolic disruption and cell death "3$. The cellular events that follow this, membrane effect are comple0 and depend on a variety of factors, such as the growth phase of the cells, the dose, and the mode of amphotericin 1 administration "4$. ome studies suggest that cell lethality is not simply a consequence of changes in permeability of mem . branes, and that formation of active o0ygen species may play a role in the lytic or lethal actions of amphotericin 1 "8, 2$.

2. Clinical manifestations of nephrotoxicity

The most restrictive adverse effect associated with amphotericin 1 therapy is its potential to induce nephroto0icity, manifested as disturbances in both glomerular and tubular. function. The clinical mani. festations usually include a9otemia, renal tubular acidosis, decreased concentrating ability of the -idney, and electrolyte disturbances such as urinary potassium wasting and hypo-alemia, and magnesium wasting and hypomagnesemia "#:$.

ris- factors. In a survey of &3 patients treated between #2&3 and #23,, at the time of the introduction of this agent, 2,( of patients developed values of 1'* e0ceeding ):: mg;#, and 8,( had serum creatinine levels greater than .#& mg;# "##$. A more recent report indicates that in almost every patient treated with amphotericin 1, the glomerular filtration rate falls appro0imately +:( within the first ) to , wee-s of therapy, stabili9es at ): to 3:( of normal and remains at this level throughout the course of treatment "#)$. %lements and <eacoc- reported an incidence of 3:( in a retrospective report conducted between #28+ and #284 "#,$. In general, the incidence in the literature ranges between &:.2:(. This variability may reflect various factors, among which are the definition used for nephroto0icity, the dose of amphotericin 1, con. comitant administration of other nephroto0ic agents, and other proposed ris- factors. In general, a9otemia is a transient occurrence that is limited to the duration of therapy= renal function usually returns to baseline after discontinuation of the drug. In many cases, cessation of therapy for a few days allows renal function to recover enough to permit administration of the full course of therapy. In rare cases, however, permanent renal damage persists after cessation of therapy, and it is not clear whether this is related to total dose or individual predisposition to to0icity. The relationship of the cumulative dose of am . photericin 1 to the development of nephroto0icity is controversial. >arlier studies suggested that greater cumulative doses of amphotericin 1 (e.g. ,.+ g! were associated with a greater ris- of nephroto0icity "#+$. This implies that the li-elihood of a rise in the serum

2.1. Azotemia The incidence of amphotericin 1.induced renal im. pairment is highly variable depending on the definition of nephroto0icity and upon the presence of underlying

creatinine concentration increases in proportion to the length of therapy. ?ur e0perience indicates that the frequency of nephroto0icity does not incre@se with e0tended therapy (6ig. )! "#&$. Ae observed patients who developed a9otemia at doses ranging from #:: mg to #.& g, with no significant increase in frequency as the cumulative dose increased. /owever, with larger cumulative doses, renal impairment may be irreversible, as reported by Ainn "#3$ who found persistent renal impairment in 88( of patients whq had received cumulative doses e0ceeding & g. everal factors influence the development of a9otemia. The frequency of dosing may be one such factor, as administration of drug on alternate days reduces the incidence of nephroto0icity "#4, #8$. Age has been suggested as a ris- factor for development of a9otemia with greater incidence in the older age group "#+, #2$. A recent case.control study revealed that higher daily doses, concomitant diuretic use, and abnormal baseline renal function are significant risfactors for amphotericin 1 nephroto0icity "):$.

glomerular filtration rate, and is temporally unrelated to a9otemia. 1arbogur et al. "),$ reported , patientswho developed an inability to concentrate the urine due to a defect in free water reabsorption even under ma0imal stimuli, indicating a tubular functional abnormality with failure of the vasopressin response in the medullary collecting tubule.

2.3. Electrolyte disturbances

>lectrolyte disorders secondary to renal wasting of potassium and magnesium are commonly encountered adverse effects in patients receiving amphotericin 1 "#:, ):$. Although hypo-alemia has been emphasi9ed in prior studies, its impact on patient management and on the course of other manifestation of amphotericin 1 nephroto0icity has not been well e0amined. In addition to its -nown systemic effects (muscle wea-ness, fatigue, cramps, rhabdomyolysis and myoglobinuria!, potassium depletion may alter renal function causing impairment of concentrating ability, urinary acidification, renal insufficiency and abnormal sodium reabsorption ")+$. It is conceivable that these effects may influence or contribute to the nephroto0icity of amphotericin 1. Appro0imately 4&( of patients will develop hypo. -alemia during the course of treatment . with am. photericin 1 ")&$. /owever, if the requirements of potassium supplementation to maintain a normal plasma level of potassium are regarded as ob5ective parameters of a potassium losing diathesis, the incidence increases to 2:( or more "#,, )3$. The maintenance of normo-alemia may be difficult and require up to ,:: m>q of potassium chloride replacement a day. These patients are often severely ill and unable to tolerate oral supplementation, so it may be necessary to administer large intravenous doses of potassium chloride over relatively long intervals (3.4 hr!, and with appropriate careful monitoring. The logistics of such continuous intravenous maintenance infusions may create problems in timely drug administration "#,$. A dose.dependent response has been proposed by some investigators, but the mechanisms by which urinary potassium wasting ta-es place are not clear. elective distal tubular epithelial to0icity seems to be, at least in part, responsible for the profound potassium wasting. Alternatively, a recent study has shown that amphotericin 1 also affects sodium flu0 in both the distal and transverse human colon, suggesting a change in sodium;potassium e0change to result in potassium loss ")4$.

2.2. Inability to concentrate the urine

Bany studies have shown that amphotericin 1 can induce a loss of concentrating ability of the -idney "##, )#, ))$. This abnormality is almost invariably present in all patients and occurs early (#.) wee-s! in the course of therapy. The impairment in concentrating ability probably reflects direct tubular to0icity

Figure 2. >stimated proportion of patients retaining normal renal function during therapy with amphotericin 1. <atients received amphotericin 1 with (C, n C #4! or without (D, n C )#! parenteral salt supplementation due to co.administration of ticarcillin. ('sed with permission from Elin Aochenschr 1987; 65: 500-6, reference "#&$!.

since it occurs in the absence of a decrease in

Bagnesium wasting has also been reported as a consequence of amphotericin 1 administration ")8, )2$. Bagnesium balance is probably decreased in all patients, but clinically relevant magnesium depletion only occurs when the urinary loss is .high and not replaced. In the study by 1arton et al., the lowest serum level and the largest fractional e0cretion of magnesium were observed by the fourth wee- of therapy, after a cumulative dose of appro0imately &:: mg, which suggested a plateau effect #)8$. This abnormality was fully reversible, evideneed by the normal serum and urinary magnesium levels measured # year after discontinuation of therapy. 1ut as in the case of potassium depletion, if the presence of magnesium depletion is evaluated not only by the serum level but also using more specific tests, the incidente of mag. nesium depletion may increase. In a recent study, a mar-ed drop in the urinary e0cretionF of magnesium occurred after a cumulative dose of only #&: mg, suggesting some degree of magnesium depletion, although serum magnesium levels remained in the low normal range ")3$. The mechanisms for the observed amphotericin 1 induced renal magnesium wasting are not clear= however, increased urinary e0cretion of magnesium despite its reduced filtered load suggests a tubular defect in magnesium reabsorption ")8$. Ahen mag. nesium and potassium wasting occur concomitantly, potassium replacement may not be successful unless magnesium deficiency is corrected first. 2.4. Renal tubular acidosis Genal Tubular acidosis can occur in patients receiving total doses of :.&.# g or more, and is generally reversible if therapy is discontinued "&$. In our e0. perience this is one of the earlier manifestation of tubular to0icity, since all patients developed an inability to acidify the urine in response to an acid load after ) wee-s of therapy and a cumulative dose of ,:: mg of amphotericin 1 ")3$. This defect appears to be a specific tubular effect of amphotericin 1, since defects in acid secretion have been demonstrated in the isolated turtle bladder and attributed to increased passive permeability of the luminal membrane to hydrogen ions ",:, ,#$, and because the impaired e0cretion of titratable acids is greater than can be accounted for by depression of glomerular filtration rate ",), ,,$. It is also thought that distal renal tubular acidosis is a contributing pathogenetic mechanism for urinary losses of potassium and magnesium ")&, ,), ,,$.

,.

Pathology

Despite the almost universal changes in renal function, histological changes associated with amphotericin 1 therapy are minimal and occur in both the glomerulus and renal tubule. Tubular damage primarily involves the distal convoluted tubule and the ascending limb of the loop of /enle ")8$. Borphologic changes include fragmentation and thic-ening of basement membranes, necrosis and vacuoli9ation of distal tubular epithelium and nephrocalcinosis "##, )#, ,,, ,+$. Hlomerular changes include calcific foci, along with hypercellularity and vacuoli9ation of smooth. muscle cells in small arteries and arterioles ")), ,,$.

+.

Mechanisms of nephrotoxicity

1efore mechanisms can be proponed to account for renal cell in5ury, the possible sites of nephron in. volvement should be identified based upon structural and functional changes ",&$. Amphotericin 1 is -nown to cause acute renal vasoconstriction and to damage preferentially the distal tubular epithelium, but the e0act mechanisms mediating its nephroto0icity have not been clearly defined. The initial event is thought to involve binding of amphotericin 1 to membrane sterols in the renal vasculature and renal epithelial cells to alter membrane permeability. This interaction may then trigger other cellular events that result in activation of second messenger systems, release of mediators or activation of renal homeostatic mechanisms. It is, therefore, possible that the membrane effect per se is not the sole factor that determines the .e0tent of change in renal function. 6urthermore, factors which interact with these secondary responses and mechanisms may modify the net effect of amphotericin 1 on renal function.

4.1. Effects on cell membranes It is generally accepted that amphotericin 1. induced in5ury to cells is due to its binding to sterols in the cell membraneI ergosterol in the case of fungal cells and cholesterol in mammalian cells "4$. This binding is more avid to ergosterol than to cholesterol, which e0plains amphotericin 1Js relatively selective to0icity to fungal cells ",3, ,4$. In the early #23:s, studies showed that polyene antibiotics induced changes in cellular permeability that resulted in the lea-age of important cellular

constituents, followed by lysis and death ",8.+)$. It

was also discovered that the to0ic effect of the drugs on cells was dependent on the presence of sterols in the cell membranes, and that addition of sterols to the growth media of certain fungi prevented the polyene induced inhibition of growth and permeability changes ",8, +,, ++$. This increased permeability has been documented in both artificial and natural membranes "+&$. It has been proposed that amphotericin 1, acting as a pseudophospholipid, interacts with sterol molecules to cause formation of aqueous pores which consist of an annulus of polyene and sterol in which the hydrophillic region of the drug molecule faces the interior of the pore (6ig. ,! "+&.+4$. Among the documented effects of amphotericin 1 on living tissues are increasing the permeability of the toad urinary bladder to urea, potassium and chloride "+8&:$, of erythrocytes and liposomes to potassium "&#, &)$, and of erythrocytes to sodium and chloride "&,, &+$. It also alters the permeability of the turtle bladder and of purified renal brush border membrane vesicles to hydrogen ",:, &&.&4$. %onsidering the renal tubular effects that are observed in clin7cal practice, it is reasonable to suggest that part or all of these effects may be e0plained by a direct effect on tubular cell membranes. In support of this suggestion is the in vivo finding that amphotericin 1 binds to sterols in most tissues, with the highest level documented in the -idney "&8$. 6urthermore, binding of amphotericin 1 to the luminal membrane of the renal tubular cells appears to be necessary for its to0ic effect on them "&3, &2$. In agreement with these suggestions is the finding of increased tubular permeability to inulin in vivo in rats following acute or chronic administration of amphotericin 1, resulting in bac-. lea- of inulin "3:$.

bac-.lea- into the interstitial space. The e0act mechanisms of the contractile responses to amphotericin 1 have not been identified. Theoretically, the drug can act either directly on the vascular smooth muscle or through release of secondary mediators. *either renal denervation nor angiotensin II receptor bloc-ade prevent the renal vasoconstriction or the reduction in glomerular filtration rate "3&$. >ndothelin does not appear to be involved in the acute responses to amphotericin 1 "33$. A role for thrombo0ane A) was suggested based upon partial inhibition of the amphotericin 1. induced vasoconstriction and reduction in glomerular filtration rate by pretreatment with ibuprofen or a thrombo0ane receptor antagonist "34$. It has also been suggested that activation of tubuloglomerular feedbac- may play a role in the acute renal effects of this compound. That hypothesis suggested. that the tubular to0icity of amphotericin 1 resulted in impaired reabsorption of sodium and chloride by the pro0imal tubule which increased distal tubular delivery of these ions, thus activating tubuloglomerular feedbac- "38$. Indirect evidence in support of a role for tubuloglomerular feedbac- was derived from studies which demonstrated inhibition of the acute renal effects of amphotericin 1 by physiological and pharmacological interventions that also bloc-ed tubuloglomerular feedbac-, namely, salt loading, and administration of furosemide, theophylline or calcium . channel bloc-ers "3).3&, 32.43$. Kater studies provided evidence against a role for tubuloglomerular feedbac- in acute amphotericin 1 nephroto0icity. It was discovered that, in contrast to its inhibition of tubuloglomerular feedbac- activity, theophylline prevented the acute renal responses to amphotericin 1 by a mechanism unrelated to adenosine receptor antagonism "44$. 6urthermore, micropuncture studies revealed that the amphotericin 1.induced reduction in single nephron glomerular filtration rate was the same irrespective of whether tubuloglomerular feedbac- was active or interrupted (by measuring glomerular filtration rate from distal and pro0imal tubular collections, respectively! "48$. That study also showed that distal tubular chloride concentrations were not increased by amphotericin 1 which indicated that the signal for tubuloglomerular feedbac- was unchanged. 6urthermore, amphotericin 1 did not

4.2. Effects on physiologic parameters

4.2.1. Acate studies

Infusions of amphotericin 1, intravenously or into the renal artery, induce short term reduction in renal blood flow and glomerular filtration rate, and an increase in renal vascular resistance, in both rats and dogs "3#3,$. The effects of short term infusions of amphotericin 1 on the renal microcirculation in rats revealed that the single nephron glomerular filtration rate was decreased by ) mechanisms (Table #!I #! a decrease in single nephron plasma flow, due to vasoconstriction of the afferent and efferent arterioles, and )! a reduction in the glomerular capillary ultrafiltration coefficient (Ef!, an effect probably mediated by mesangial cell contraction "3+$. <revious micropuncture studies demonstrated a similar vasocon. striction of the afferent arteriole but also an increased permeability of the tubular epithelium to inulin "3:$. Thus, the reduction in glomerular filtration rate after acute amphotericin 1 infusions can be attributed to contraction of afferent and efferent arteriolar smooth muscle cells and of glomerular mesangial cells, as well as increased tubular permeability with

figurr 3 Proposed partial

model for the mB!induced pore in the cell membrane. "he drug acts as a counterfeit phospholipid# $ith te C. lyetroxyt %& carboxyl# and %1' mycosamine groups situated at the membrane!$ater interface# and the %# to % 14 and C20 to asigned peral( $ithin the membrane. The heptaene chain will seek a hydr ph bic en!ir nment while the hydr "yl groups see) a hydrohibc en!ir nment. Th#s, a cylindrical p re will be $ rmed, the inner wall $ which c nsists $ the y r datylsnsa*ted crbon chains $ the %m& m lec#les, and #ter wall $ which is $ rmed by the heptaene chains $ the moscules

appear to increase the sensitivity of the tubulog . lomerular feedbac- mechanism. In vitro e0periments, on the other hand, demonstrated a direct vasoconstrictor action of amphotericin 1 in perfused afferent arterioles and isometrically contracting rings of rabbit aorta and renal artery, effects which were prevented in %a ''.free medium and by %a' L channel bloc-ers or theophylline. Thus, amphotericin 1.7nduced reductions in renal blood flow or glomerular filtration rate are not secondary to activation of tubuloglomerular feedbac-, but due to a direct vasoconstrictor effect. These results are consistent with our findings in cultured glomerular mesangial cells where am. photericin 1 caused a concentration.dependent in. crease in intracellular calcium levels ("%a ' which was almost completely inhibited when either %a' or *a- ions were omitted from the cell medium (6ig. +! "42$. Diltia9em (): p..B! also suppressed the rise in "%a ('$i seen with amphotericin 1. These results indicated that the reduction in E, observed in vivo was most li-ely due to contraction of mesangial cells. These two studies "48, 42$, therefore, suggest that the contractile effects of amphotericin 1 in the nephron are probably due to a direct interaction with cell membranes, leading to formation of pores. ?ne possibility is that these pores result in entry of *a' ions into the cells down their electrochemical gradient, which leads to depolari9ation. induced opening of voltage.dependent calcium channels and contraction. 6inally, recent studies suggest a protective effect of pento0iphylline, a vascular decongestant and antagonist of tumor necrosis factor.a and interleu-in.#a, against amphotericin 1.induced acute and chronic nephroto0icity, suggesting a role for these factors in the renal effects of the drug "8:, 8#$.

Figure 4. ) ncentrati n dependent increase in intracell#lar

4.2.2. Chronic studies

Animal models of chronic nephroto0icity have also

calci#m le!els in c#lt#red *l mer#lar mesan*ial cells +%, and its inhibiti n by rem !al $ )a '' and -a i ns $r m the medi#m +-)a and --a, respecti!ely,, and by additi n $ (0 aB diltia.em +/ilt, +&,. +0sed with permissi n $r m 1#r 2 3harmac l 199(; ((6: 79-85, re$erence 4795,.

shown that certain interventions can modify the ne. phroto0icity of amphotericin 1. Gats co.treated with sodium bicarbonate sustain smaller reductions in glomerular filtration rate compared with control rats treated with amphotericin 1 alone for , wee-s ",)$. ?ral *a%# supplementation also attenuates the de. . crease in glomerular filtration rate and the elevation in renovascular resistance induced by daily administration of amphotericin 1 over , wee-s "4,, 4&$. In addition, renal impairment following a 4 day course of amphotericin 1 in rats was less severe when theophylline was co.administered "43$. These interventions also att enuat e the acut e renal responses to am photericin 1, suggesting that similar mechanisms contribute to its chronic nephroto0icity. imilar logic would suggest that salt supplementation and theophylline are protecting the -idney by a mechanism unrelated to tubuloglomerular feedbac-. It is, however, possible that the latter does contribute to amphotericin 1 nephroto0icity but only at later stages of therapy, when severe damage to the tubules may have ta-en place. Interestingly, the protection by salt loading is associated with less concentrations of drug in the -idney despite similar concentrations in plasma and liver tissue "4,$. This raises an alternat7ve possible mechanism of protection by salt loading involving a pharmaco-inetic interaction with amphotericin 1 which limits its upta-e into the -idney. tudies on the effects of chronic administration of calcium channel bloc-ers on chronic amphotericin 1 i nduced neph rot o0 i ci t y have been di scord ant . *ifedipine does not offer a significant protective effect "8)$, but diltia9em blunts the increase in serum creatinine and the decreases in glomerular filtration rate and renal plasma flow "4+$. It is possible .that these differences relate to the heterogeneity of calcium channels and the differential activity of calcium channel bloc-ers on them. /owever, no specific studies have addressed this question. A recent study demonstrated that coadministration of &.flucytosine with amphotericin 1, which is com. monly used clinically to obtain a synergistic antifungal effect, protects against acute and chronic nephroto0icity "8,$. The mechanisms by which flucytosine influences the renal response to amphotericin 1 are not clear but may relate to (#! its administration in :.2( *a%#, which itself is protective, ()! a renal vasodilator effect of flucytosine that antagoni9es amphotericin 1.induced vasoconstriction, and (,! reduction in renal upta-e of amphotericin 1 "8,$. %ell death induced by amphotericin 1 in the medullary thic- ascending limb is prevented by ouabain "8+$. A

reasonable e0planation for this observation is that ouabain, by inhibiting transport, decreases, the o0ygen demand of that area of the nephron, which has a limited o0ygen supply to start with. It is conceivable that amphotericin 1.induced renal vas oconstriction and ischemia to this section of the nephron would enhance the cell death produced by a direct to0ic action of amphotericin 1 on those cells. Thus, any maneuver that improves renal perfusion, or decreases o0ygen demand, would be e0pected to be protective. This may e0plain the salutary effect on amphotericin 1 nephroto0icity of s@lt loading, theophylline, and calcium channel bloc-ers, all of which improve renal blood flow, and of furosemide, which, in addition, inhibits transport.

+. Measures to reduce nephrotoxicity

Despite being considered one of the most to0ic antimicrobial drugs in use today "8&$, amphotericin 1 remains the drug of choice for otherwise uniformly fatal systemic fungal infections "+, &$. %onsequently, it will remain in use despite the predictable occurrence of severe systemic and renal to0icity. Therefore, therapeutic interventions that decrease amphotericin 1 to0icity assume critical importance. Among the early interventions that were e0amined is administration of mannitol. >arly studies suggested a protective effect of mannitol in dogs and renal transplant recipients "83M 88$. 'nfortunately, these were either case.reports or poorly controlled studies. Kater reports failed to detect any protective effect of mannitol in dogs, and ascribed the earlier findings to the lower doses of amphotericin 1 used "82$. In addition, a small controlled trial of mannitol coadministration in humans failed to document any beneficia! effect "))$. In addition to the protective interventions mentioned in the previous section, animal e0periments have provided evidence for a protective effect of dopamine or dopamine agonists on the acute and chronic renal effects of amphotericin 1 "2:.2)$. Theoretically, all these interventions may be applicable in the clinical setting, but few have actually been studied. 6urthermore, there are some practical limitations to their use. 6or e0ample, the duration of protection conferred by furosemide is brief, being confined to the time furosemide is present in the renal tubule. 6urosemide, moreover, may e0acerbate electrolyte imbalance by causing sodium and potassium depletion, which, if not adequately monitored and replaced, may potentiate amphotericin 1.induced ne. phroto0icity. 6inally, aminophylline is not a totally innocuous drug. In contrast, manipulation of sodium

status offers a simple intervention that can be readily introduced into clinical practice "38$. Alternatively the use of a different formulation of the drug which may alter its delivery to cells and reduce its interaction with membrane sterols of -idney cells may be beneficial, and that is the principal goal of the new series of preparations that are either lipid comple0es of the drug or liposomal forms of it.

course of amphotericin 1 for persistent fever of un-nown origin. ?nly two of ): patients (#:(! developed mild renal dysfunction which, however, did not necessitate interruption of therapy. The full course of high.dose amphotericin 1 was successfully completed in all p@tients, including four with mild pretreatment renal impairment. The question of the influence of salt supplementation was finally addressed using a prospective randomi9ed placebo controlled trial of the influence of salt supplementation on the course of renal function during therapy with amphotericin 1 ")3$. Amphotericin 1 administration was preceded by # liter of either :.2( saline or &( de0trose in water administered i.v. over + hr. Ahile the mean serum creatinine increased over time in the de0trose group, it remained unchanged in the saline group. imilarly, creatinine clearance decreased in the de0trose group, but remained unchanged in the saline group. The beneficial effect of salt loading, however occurred at the e0pense of greater hypo-alemia, with the saline group requiring significantly higher amounts of potassium supplementation to maintain a normal serum E level (6ig. &!. Therefore, based upon these studies, it is reasonable to recommend routine salt supplementation with administration of amphotericin 1, with special attention being paid to maintaining potassium balance. 5.1.1. Other formulations: liposomes The wide ranging to0icity of amphotericin 1 led to the development of new formulations of the drug, whereby it was either intercalated into liposomes (K.amphotericin 1! or comple0ed to phospholipids. In the last decade, these preparations have been e0amined e0tensively in in vitro, whole animal and clinical studies "2+.##2$. Any comparison of conventional amphotericin 1 with a new formulation of the drug should address the following questionsI #! do the two formulations have the same or different actionsN )! if they have the same action, what is the dose ratio for their antifungal and to0ic effects, especially nephroto0icityN and ,! is there a selective advantage in the dose ratios indicating a wider therapeutic margin, i.e., is the dose ratio Kamphotericin 1 ;amphotericin 1 lower for the an. tifungal effect compared to the dose ratio K.amphotericin 1 ;amphotericin 1 for the nephroto0ic effectN The collective evidence in the literature suggests that K. amphotericin 1 and conventional amphotericin 1 have a similar action on fungal and mammalian cells. Oery few studies, however, have established a dose ratio for antifungal and nephroto0ic effects. In

+. 1.

, al t

suppl em en tati on

tudies in animal models which demonstrate a renal protective effect of salt loading on amphotericin 1induced nephroto0icity have provided a rational basis to evaluate this simple intervention in patients. %linical evidence supporting the ability of sodium loading to attenuate amphotericin 1. induced nephroto0icity is derived from three sourcesI case reports, retrospective studies and prospective studies. ?ne of the earliest case reports was by 1utler and colleagues who reported a patient in whom a low sodium diet (2 m>q;d! e0acerbated renal dysfunction, increased urinary sodium loss, and caused postural hypotension "##$. Administration of supplemental oral sodium chioride promptly reversed the defect within #) hours. These abnormalities were confirmed on rechallenge during treatment, but were absent #, months after completion of amphotericin 1 therapy. In a more recent report, & patients receiving amphotericin 1, in whom several salt conserving states were identified, including dietary salt restriction, vomiting, diuretic therapy, AddisonJs disease and cirrhosis with ascites, susta7ned increases in 1'* and serum creatinine levels within 3 to #) days after starting amphotericin 1 "2,$. 6our to #) days after liberali9ation of dietary sodium inta-e, administering intraven. ous saline, and;or discontinuation of diuretic therapy, renal function improved in all patients. Improvement was sustained and the full course of amphotericin 1 was successfully completed after a brief interruption (rangeI # to & days!, without permanent renal impairment. A retrospective study revealed that only two of #4 patients (#)(! receiving ticarcillin (with its obligatory sodium load of #&: m>q;day! had a nephroto0ic response to amphotericin 1, compared with #+ of )# patients (34(! not receiving ticarcillin (6ig. )! "#&$. Anecdotally, withdrawal of ticarcillin in + patients led to deterioration of renal function over a one.weeperiod. In a companion study, the benefit of routine intravenous saline (# K of :.2( saline! was assessed prospectively in leu-emic patients receiving a )8.day

lethality of the preparation, which usually is related to the acute infusion of the drug and may not necessarily relate to other manifestations of to0icity, especially nephroto0icity "2&, 24.22$. 6inally, efficacy studies are complicated by difficulties in the diagnosis of fungal infection= the underlying clinical condition of the patients, and frequent concomitant use of other nephroto0ic agents. Aith this in mind, a review of the literature reveals some variability in the results and conclusions. Galph et al. demonstrated that K.amphotericin 1 is generally less active than amphotericin 1 on yeast cells, and has a slower onset of action "#::$. The authors suggest that K.amphotericin 1 acts as a reservoir for free amphotericin 1 which is the active moiety. ?thers found either an equivalent or ,.+ fold less efficacy for a lipid.comple0ed form of amphotericin 1 "2&, 28, #:#$. These differences may be attributed to the different preparations used and;or to the different strains of fungi e0amined. In one of the fe w studies which calculated a concentration ratio for the actions of K.amphotericin 1 and conventional amphotericin 1 on mammalian and fungal cells, Puliano et al. compared the in.vitro to0icity of amphotericin 1 and K. amphotericin 1 to %andida albicans and mammalian erythrocytes "23$. Ahile the two formulations were equipotent in their effects on ion flu0es in yeast cells (indicating formation of membrane pores!, only amphotericin 1 induced such an effect in erythrocytes, despite achieving concentrations of K.amphotericin 1 that were #: to ):.fold higher than those of amphotericin 1. The time required to achieve this effect in fungi was the same for the two formulations, which suggested that Kamphotericin 1 did not constitute a slow.release form of the drug. The reason for the reduced to0icity of K.amphotericin 1 is proposed to be a preferential transfer of amphotericin 1 from liposomes to fungal cells compared with its transfer to mammalian cells. 6ew studies have compared the antifungal and to0ic effects of the two formulations to confirm a wider therapeutic indeQ. The same authors, however, demonstrated a greater to0icity of amphotericin 1, compared with K.amphotericin 1, to -idney epithelial cell structures (using KK%<E# cells! after short e0posure times () hr!, with different formulations of K. amphotericin 1 e0hibiting different >%&:Js, the most potent having an >%&: #, times that of amphotericin 1 "#:)$. Ta-en in concert with their previous study "#:#$, this indicates that the to0ic concentration ratio of K.amphotericin 1 ;amphotericin 1 (#,.):I#! is higher than its antifungal concentration ratio (#I#!. In contrast, chronic e0posure of KK%<E# cells to Kamphotericin 1 (#.) days!, which is more representa.

Figure 5. 6er#m 7 le!els +%, and 7 s#pplements *i!en t maintain ser#m 7 le!els at r ab ye 8 mm l9: +&, in patients recei!in* %m& with either 1 liter $ 0.9; -a)1 +0, r 1 liter $ 5; de"tr se in water +%,. - tice the di$$ic#lty in maintainin* ser#m 7 le!els despite si*ni$icantly hi*her am #nts $ s#pplements in the $ rmer *r #p. <: 3 = 0.05 c mpared with baseline. +0sed with permissi n $r m 7idney :nt 1991; >0: 80(-808, re$erence 4(65,.

m o st , o nl y o ne a s pe c t o f t h e a c t i vi t y o r o nl y o ne formulation was studied. Another difficulty in interpreting these studies is that there is not one single formulation being assessed but several, prepared either by the investigators themselves or by pharmaceutical companies. Thus, comparisons between studies be.come more difficult, and generali9ations should be made with care. In support of this conclusion is the demonstration .that the fungicidal activity of K.amphotericin 1 is influenced by several properties of the liposomes including lipid composition, physical si9e, the molar ratio of lipids, and the presence or absence o f s t e ro ls " 2+ . 2 3 $. 6 ur t h e r m or e , t he t e s t s u se d t o assess in vivo to0icity have rarely e0amined renal function adequately, and many rely on assessing the

tive of what occurs clinically, resulted in profound to0ic effects at concentrations similar to those of amphotericin 1 (K.amphotericin 1 ;amphotericin 1 C #I#!, manifested by changes in cellular transpon processes and in morphology "#:)$. This finding raises questions as to the applicability . and relevance of results derived from short.term in.vitro e0periments to whole animal or clinical situations. ?ther in vitro studies in primary cultures of pro0imal tubular cells demonstrated increased E efflu0 and KD/ release with amphotericin 1 but not. K.amphotericin 1 despite achieving + to 8 foid higher concentrations of the latter "#:,$. 'nfortunately, tifungal activity was not assessed in this study and no dose ratio can be calculated. The acute nephroto0icity of the two formulations was e0amined in rabbits where amphotericin 1 induced a fall in glomerular filtration rate and a rice in urinary sodium and potassium e0cretion rates, while. K. amphotericin 1, at ).& times the conventional dose, did not affect these parameters "#:+$. /ere again, antifungal activity was not assessed. 'ne0pectedly, however, and in contrast to amphotericin 1, K.amphotericin 1 increased the e0cretion of *acetylglucosaminidase in these animals. The authors suggested that increased delivery of amphotericin 1 to the cells by the liposomes led to an interaction with lysosomes and provided an additional mechanism of in5ury. In support of this conclusion is the finding that the tubular to0icity induced by repeated administration of amphotericin 1 and K.amphotericin 1 over & days was similar when a high enough dose of K. amphotericin 1 ().+ times that of amphotericin 1! was used "#:&$. . Animal studies suggest that K.amphotericin 1 is effective in the treatment of fungal infections, but usually requires higher doses than amphotericin 1 "2+, 24, 28, #:3, #:4, ##3, ##2$. The lac- of concomitant assessment of renal function in many of these studies ma-es it impossible to determine a dose ratio, al. though most report that the drug was well.tolerated. . imilar findings have been reported in pat7ents, with several studies showing that those who failed to respond to conventional amphotericin 1, or those who developed nephroto0icity on it, were either complete or partial responders to K.amphotericin 1, and did not sustain deterioration in renal function "#:8.##&, ##4, ##8$. The administration of K.amphotericin 1 was also devoid of other adverse effects associated with amphotericin 1, and was generally well.tolerated compared with previous studies using amphotericin 1. It is clear from in vitro and animal studies that newer formulations of amphotericin 1 have antifungal and to0ic effects similar to those of amphotericin 1.
an.

1oth, however, occur at doses or concentrations higher than those of conventional amphotericin 1. 6ew studies have unequivocally demonstrated a wider ther@peutic margin for any new formulation. The results from initial clinical studies are encouraging, but none of these reports can be considered as definitive evidence for a wider therapeutic inde0. The ma5or reason for this is the lac- of controlled prospective randomi9ed comparisons of K.amphotericin 1 and conventional amphotericin 1 with regard to both antifungal and nephroto0ic effects. 6urthermore, a longer follow.up may be necessary to ensure that the finding of lower to0icity with K. amphotericin 1 is not associated with greater recurrence of infection.

-. %linical use
-.1. .eneral underlying condition of the patient
The indication for amphotericin 1 is the presence of proved or suspected systemic fungal disease. As previously mentioned, patients who receive amphotericin 1 are usually severely ill, with some degree of malnutrition, multiple organ failure and life threatening infections. The clinical condition of these patients may, therefore, confer an increased ris- oI nephroto0icity. ?nce the decision to implement am photericin 1 has been made, an algorithm can be used to optimi9e therapy (6ig. 3! "38$. The first step is assess the sodium status of the patient, and to correc overt sodium depletion. It is important to reali9e tha milder sodium depleted states which are not apparent can substantially enhance the nephroto0ic potential of amphotericin 1. It is, therefore, importan to assess whether the patient can tolerate sodiun supplementation in addition to a normal salt inta-e ?therwise healthy sub5ects usually tolerate a supple ment of #&: m>q;d over . and abo y e the norma sodium inta-e of #&: to ):: m>q;d without difficulty Increased sodium inta-e, however, may e0acerban cardiac failure, cirrhosis with ascites, or renal failure Another factor to consider is whether the patien will receive sodium supplementation as a consequence of concomitant antibiotic therapy (e.g., Ahen the opportunity to choose among several anti biotics arises, the alternative with the highest obligat ory sodium load should be selected whenever possible 6inally, it is prudent to chec- for the presence o potassium and magnesium deficits prior to therap since amphotericin 1 will invariably cause loss these electrolytes during therapy. %orrection of thes abnormal7ties, before or concomitantly with the .star

of therapy, should delay or avoid the early develop . ment of electrolyte disturbances and possible additional to0icity (e.g., arrhythmia, rhabdomyolysis! that sometimes necessitate the early discontinuation of therapy. 6.2. Amphotericin administration

cated, amphotericin 1 is given between two ,:.min infusions of :.& K normal saline intravenously. This amount of supplementation is based on empiric ob. servations, and further studies are needed to ascertain whether lower amounts confer an equivalent degree of protection. If vomiting occurs, sodium supple . mentation may be increased.

Amphotericin 1 therapy should be started with a low dose, gradually escalating to a full therapeutic dose according to patient tolerance. Ae advocate adminis. tration of amphotericin 1 as a four.hour infusion between doses of ticarcillin. If ticarcillin is not indi.

6.!. Concomitant use of nephrotoxic dru"s If amphotericin 1 is to be used in con5unction with another nephroto0ic agent, several measures can be

ta-en in order to minimi9e the potential synergistic to0icity of amphotericin 1. 6or e0ample, if an amino. glycoside or cyclosporine is to be used, monitoring of their serum concentrations will help avoid to0ic levels. It is also imperative to evaluate electrolyte losses closely and to be aggressive in their replacement. ?ne approach is to give the minimum requirements either by diet or supplements plus the urinary losses, with a close follow up of the blood levels. %yclosporine A is commonly used with amphotericin 1, in which case it is doubly important to maintain the patient in a well hydrated condition, since cyclosporine A nephroto0icity may be e0acerbated by dehydration "#):$, and to follow magnesium levels closely since both drugs cause hypomagnesemia.

therapy with amphotericin 1. /owever if renal function continues to deteriorate, or if the rate of deterioration is rapid, temporary discontinuation of amphotericin 1 therapy may be required. Therapy can be resumed in rehydrated patients when serum creatinine concentrations begin to return toward baseline values.

7. %onclusion
Amphotericin 1 remains the best agent to eradicate severe disseminated fungal infections. *ephroto0icity is the most limiting adverse effect associated with its use, frequently requiring discontinuation of therapy or prolongation of hospital stay. It commonly presents as a9otemia and a fall in glomerular filtration rate, along with several abnormalities in tubular function. The mechanisms of nephroto0icity are li-ely to involve direct vasoconstrictor effects and direct to0ic effects on tubular cells, probably related to the actions of the drug on cell membranes to alter their permeability. To date, the only clinically proven intervention that lowers the incidence and severity of nephroto0icity is salt supplementation, and it should probably be used routinely in patients who can tolerate it. There are suggestions that liposomal or lipid.comple0ed formulations of amphotericin 1 may have a wider therapeutic inde0, but more studies are required to support this contention.

-.4. Potassium and magnesium supplementation

'rinary potassium and magnesium losses are antici . pated consequences of amphotericin 1 therapy. ome of the losses can be compensated with increased dietary inta-e, while others will require oral or intravenous replacement. It should be recogni9ed that the serum levels of these ions do not necessarily correlate with the total deficit, as the plasma levels tend to be conserved while cellular stores are becoming depleted. In general, potassium and magnesium supplements should be given to all patients and the amounts increased if the potassium level falls below ,.& m>q ;# or the magnesium level falls below #.3 m>q ;I, with either dietary or pharmacological supplementation. Amiloride, in low doses, is an alternative therapy in patients who need high dose intravenous potassium replacement "#)#$.

c)no$ledgment
Th e re sea rc h wo r - p re se nt ed in th is cha pt er wa s supported by 'nited tates <ublic /ealth ervice . grant HB+,)3,.

-.+. /ollo$!up
?nce therapy has been established, plasma levels of creatinine, potassium and magnesium should be assessed periodically. It is also recommended to have a complete blood count, electro%ardiogram, and mea.sures of serum transaminase levels, creatinine clearance and urinary electrolyte e0cretion rates, periodically. These requirements will differ among patients, and the frequency of measurements should depend on the individualJs condition. In patients with mild renal dysfunction prior to amphotericin 1 therapy, sodium supplementation has proved to be safe and effective. In patients not receiving sodium supplementation who develop renal impairment during amphotericin 1 therapy, initiation of sodium supplementation may permit continued

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