Pharmacology of agents used in bone marrow transplant conditioning regimens.

241

Critical Reviews in Oncology/Hemaiology. 1992; 13:241-270 0 1992 Elsevier Science Publishers B.V. All rigths reserved. 1040-8428/92/$5.00

ONCHEM 00036

Pharmacology

of agents used in bone marrow transplant regimens

conditioning

Valerie J. Wiebe”, Brian R. Smithb, Michael W. DeGregorio” and Joel M. Rappeportb “The University

of Texas Health Science Center, San Antonio, TX, USA and “the Sections of Hematology, Department Departments of Laboratory Medicine and Pediatrics, Yale University. New Haven, CT, USA

of Medicine,

and the

(Accepted 27 July 1992)

Correspondence to: USA.

242

I.

Introduction .......................................................

II.

Hematopoiesis

III.

Agents with predominant antihematopoietic

IV.

Busulfan and dimethylbusulfan......................................... A. Pharmacology and pharmacokinetics. .................................. B. Immunosuppression .............................................. C. Antihematopoietic activity ......................................... D. Antineoplastic activity ............................................ E. Toxicity.. .....................................................

245 245 246 246 247 247

V.

Carmustine ............................................... A. Pharmacology and pharmacokinetics ........................ B. Immunosuppression .................................... C. Antihematopoetic activity ................................ D. Antineoplastic activity .................................. E. Toxicity .............................................

247 248 248 248 248 249

VI.

Melphalan ....................... A. Pharmacology and pharmacokinetics B. Immunosuppressive activity ....... C. Antihematopoietic activity ........ D. Antitumor activity .............. E. Toxicity ......................

249 249 249 350 250 250

......................................................

244

activity. .........................

., .... .

.., ... .. ... ...

..

. ..

...... .

245

VII.

Radiation . A. Total body irradiation (TBI) B. Immunosuppression C. Antihematopoietic activity D. Antineoplastic activity E. Radiation-induced toxicity

250 250 250 251 251 251

VIII.

‘Nonablative’ antineoplastic agents ...................................... A. Cytosine arabinoside (Ara-C) ....................................... B. Thiotepa ....................................................... C. Etoposide (VP-16) and teniposide (VM-26) .............................

252 252 253 254

IX.

Immunosuppressive agents . . A. Total lymphoid irradiation (TLl) B. Cyclophosphamide

256 256 257

........................... ........................... ...........................

Dr. Joel M. Rappeport, Department of Medicine, Yale University, 333 Cedar Street, P.O. Box 3333, New Haven CT, 06510,

242 C. Cyclosporine A ................................................. D. Procarbazine .................................................... E. Antithymocyte and antilymphocyte immunoglobulins

.....................

258 259 260

X.

Monoclonal antibodies (anti-LFA) ....................................... A. Additive toxicities and drug interactions ................................ 1. Additive toxicities ............................................. 2. Drug interactions ..............................................

261 261 261 261

XI.

Discussion . . . . . . . . . . . . . . . .._._.............................._......

262

References................................................................

Disease state, donor histocompatibility, effective immunosuppression, antineoplastic activity and creation of physiological hematopoietic space are all key determinants of succesful bone marrow transplantation (BMT). Preparative BMT regimens have evolved from the use of single fraction total body irradiation (TBI) to elaborate combinations of multidrugs and fractionated TBI regimens. Many of these newly developed conditioning regiments have increased engraftment rates and curative potential. The purpose of this review is to critically examine the pharmacologic behavior of the most commonly used agents in bone marrow transplantation conditioning regimens. The known immunosuppressive, antihematopoietic and antineoplastic properties of each agent are discussed. In addition, the pharmacoki-

265

netics and toxicity profiles are evaluated. A thorough understanding of the pharmacology of agents used in BMT may aid in the design of patient specific conditioning regimens. I. Introduction Conditioning regiments play a major role in the ability to achieve and sustain successful engraftment following bone marrow transplantation (BMT). Optimal conditioning of a bone marrow transplant recipient requires both antihematopoietic and immunosuppressive effects in order to minimize the risk of graft rejection and recurrence of the underlying disease. Adequate antihematopoietic and immunosuppressive activity may

TABLE I Disease specific pharmacologic requirements for BMT Disease state

Antihematopoietic

Allogenic MHC-matched BMT: Aplastic anemia Leukemia/lymphoma KIDS Non-SCIDS genetic diseases’

_ +++ _ +++

++ ++ _ ++

+++ -

Allogenic MHC-mismatched BMT: Aplastic anemia Leukemia/lymphoma SCIDS Non-SUDS genetic diseases*

_ +++ _ +++

+++ ++ +/++

_ +++ _ _

Syngenic BMT: Aplastic anemia Leukemia Lymphoma

_ +++ _

+/_ _

+t+ +tt

Autologous BMT: Leukemia Solid tumors/lymphoma

++t _

_ _

+++ +++

Required (+++, ++, +); unclear (+I-); not required (-). *Including Wiskott-Aldrich syndrome, osteopetrosis, thalassemia, storage diseases etc.

Immunosuppressive

Antineoplastic

243

require multidrug regimens. Agents incorporated into these regimens must be specifically selected based on the patients disease state and type of BMT. Diseases treated with BMT have included an array of immunologic and hematologic disorders in addition to a variety of neoplastic diseases. Each disease requires varying degrees of antihematopoietic, immunosuppressive and antineoplastic activity produced by a conditioning regimen (Table 1). The properties required of a conditioning regimen are also dependent on the type of BMT. Typically, there are four types of transplantation based on the histocompatability of the donor and patient. These include syngeneic (identical twin), autologous (the patients own marrow). HLA-matched ‘allogeneic’ (either genotypically HLA identical siblings, phenotypically HLA identical family members or, phenotypically HLA identical unrelated donors) and HLAmismatched allogeneic BMTs. In addition to these considerations of disease state and type of BMT, other factors also contribute to the efficacy of preparatory regimens and should be evaluated prior to the design of BMT conditioning regimens. In particular, pharmacologic factors such as cell cycle specificity, pharmacodynamics, pharmacokinetics, drug interactions and overlapping toxicities of agents must be considered in the design of conditioning regimens. Ideal agents should have predictable systemic absorpTABLE

tion, and adequate distribution and penetration into tissue sanctuaries that may harbor neoplastic cells when indicated. In addition, the parent compound and its active metabolites should have sufficiently short halflives so that cytotoxic concentrations of drug are not present during transplantation. Rates of metabolism, elimination of active species, and overlapping toxicities of active agents within regimens may all influence the efficacy of conditioning regimens and ultimately engraftment and survival rates. Some of these considerations are listed in Table 2. Although the pharmacology of conventional doses has been evaluated, less is known about many conditioning agents when used in higher doses typical of BMT conditioning regimens. Although BMT conditioning regimens incorporating antihematopoietc agents were at one time considered to be 100% ablative, recent evidence indicates that ablation may not always be complete. The presistance of host-derived stem cells following conditioning has been observed following both single-agent and multidrug conditioning regimens. Persistance of host immune factors may also be involved in immunologic rejection of donor marrow. In addition, ‘unexpected’ residual drug present at BMT may also contribute to graft rejection. New approaches to allogeneic BMT, including the use of matched unrelated donors, may require enhanced immunosuppressive activity of conditioning regimens.

2

Pharmacologic

parameters

of agents

used in conditioning

Dw

Dose range*

Ara-C

l-3 g/m’

Half-life**

Site of metabolism

Activity of metabolites

CNS”’ penetration

Protein binding

a: 8-16 min

Liver, kidney

Active

622%

13%

j?: 1.5-12days

?

Inactive

‘7

‘>

a: 1-3 hrs

Liver

Unknown

>80%

7%

Liver

Active

15570%

>90%

Liver

Active

95%

Liver

Active

90%

‘?

,8: IO-15 hrs

Liver

Active

13.0%

>90%

a: 7 min /R 2-3 hrs

Liver

Active

>80%

10%

/Y:2-3 hrs

II-31 ATG [41

15 mgkg

Busulfan

1 w/k

B: 2-I hrs

[51 Carmustine

800 mg/m’

[681 Cyclophosphamide

5&60 mg/kg

[9. 101 Cyclosporine [ll. 121 Etoposide [13-151 Melphalan [I6191 Procarbazine [20, 211 Teniposide [22, 231 Thiotepa [2&28]

regimens

6 mg/kg 30 mg/kg 140-180

mg/mz

150 mg 30&750 45405

mg/m’ mg/m’

a: 32 min j?: 45 hrs /I: 0.75-8.0 a: B: a: B: a: B: j?:

hrs

l-3 hrs 46 hrs 0.1-0.5 hrs 228 hrs 8810 min 27-65 min 7 min

*Doses administered during pharmacokinetic trials; Ara-C: infusion over 3 hrs, twice daily x 6 days; Busulfan: oral every 6 h x 4 days; Carmustine: bolus over 10 min x 1-2; Cyclophosphamide: infusion over l-2 hrs/day x 24 days: Etoposide: infusion over 4 h x 1: Melphalan: bolus over 5 min x 1; Teniposide: continuous infusion over 72 hrs; Thiotepa: bolus q day x 3 days. “Pharmacokinetics vary with dose and rate of administration.“’ % CNS Penetration; CSF concentration/ plasma concentration x 100.

244

Inter-patient variability in pharmacokinetics (in absorption, clearance, sensitivity etc.) may also account for the success versus failure of BMT. Conditioning regimens designed to incorporate patient specific variables in pharmacokinetics and drug sensitivity, may improve the engraftment rates. This article reviews the pharmacologic information of agents commonly employed in conditioning regimens. Although agents will be referred to individually, single-agent conditioning of patients is generally insufficient to achieve the multiple properties required of conditioning regimens and combination therapy is usually necessary for the optimal conditioning of the BMT recipient. The pharmacology, kinetics, antihematopoietic, immunosuppressive, antineoplastic activity and toxicity of agents will be addressed.

of cells is under the regulation of many influences including feedback mechanisms and growth stimulating factors. Pluripotential stem cells have little cell cycle activity and differ in their susceptibility to cytotoxic agents. The commitment to differentiate in hematopoietic progenitors is associated with increased cell cycle activity which increases a cell’s susceptibility to cell cycle specific cytotoxic agents. Stem cells differ in their self-renewal capacity, and presumably only a few pleuripotential stem cell clones are required to maintain or establish hematopoiesis. Pluripotent hematopoietic stem cells may migrate out of the bone marrow into the peripheral circulation [29]. Stem cells, including those given by bone marrow transplantation, migrate back into the bone marrow by crossing the marrow-blood barrier [30]. Resettlement of stem cells within the bone marrow is dependent on sufficient ‘space’ (i.e., a physiological niche) for cells to grow and proliferate [3 1,321. Ablation of hematopoietic pleuripotential cells in order to create ‘space’ for newly transplanted cells may therefore be required in many conditioning regimens. Since some neoplastic diseases, such as chronic myelogenous leukemia (CML), involve the stem cell population rather than the more mature differentiated progeny, a key factor in eradicating CML and some myelo-

II. Hematopoiesis Differentation of hematopoietic progenitor cells occurs primarily in the bone marrow, with the exception of T-cell differentation which occurs predominantly in the thymus. Within the bone marrow, stem cells must commit themselves to one of several differentiation pathways (erythropoietic, granulopoietic, megakaryocytic pathways) or undergo self-renewal. Differentiation

TABLE 3 Review of major toxicities Agent

Gastrointestinal

Pulmonary

Cardiac

Neurologic

Hepatic

Renal

Other

Ara-C

N, V, D, M

Edema

Cardiomegaly

LFT. VOD

SIADH

DE, 0, FV

Edema

Tachycardia

PN, cerebellar dysfunction ~

LFT

Azotemia

ATG

Azotemia

-

Hepatitis VOD B, LFT

FV, DE, HT, serum sickness, anaphylaxis Addison’s +Lupus‘like’ Syndrome HT

SIADH, HC

DE

CD. Seizures

B

Azotemia

P, hirsutism Low Mg” DE

B, LFT

HC

FV, DE. MA, HT

LFT

_

LFT

Azotemia

DE 0, DE 0. E, FV HT. DE

LFT

_

Busulfan

N, V, D, M

Fibrosis

Carmustine

N, V, D, M

Fibrosis. IP

Tachycardia

CD

Cyclophosphamide Cyclosporine

N V, A

Fibrosis

Myocardopathy

Dimethyl busulfan Etoposide

N, V, D, M

IP

N, V. M

Obstructive disease

Tachycardia

CD

Melphalan Procarbazine Radiation Teniposide

N, V, D, M, A N V, D N, V, D, M M

_

CD, PN _ -

Thiotepa

N, V, D, M, A

HS IP Bronchospasms _

CD

B. VOD

N, A

VOD

Pericarditis Tachycardia

DE, hyperuricemia

A, anorexia; B, elevated bilirubin; CD, CNS dysfunctions; D, diarrhea; DE, dermatitis; FV, fever, HC, hemorrhage cystitis; HS, hypersensitivity reactions; HT, hypotension; IP, interstitial pneumonitis; LFT, elevated liver function tests; M, Mucositis; MA, metabolic acidosis; N, nausea, 0, ophthalmic, P, pancreatic abnormalities; PN, peripheral neuropathies; SIADH. syndrome of inappropriate antidiuretic hormone; V, vomiting.

245

dysplastic syndromes, as well as those diseases involving more differentiated progeny may require the complete ablation of all neoplastic stem cell clones capable of self renewal. Failure to completely ablate the remaining neoplastic stem cell clones capable of self-renewal, may result in a relapse of the disease. Similarly, stem cells with abnormal genes, such as in thalassemia, must be completely eliminated in order to prevent recurrence of disease. 111. Agents with predominant antihematopoietic

activity

Although not all diseases treated with BMT require antihematopoietic activity in the conditioning regimen (Table l), the majority of diseases do require some degree of marrow ablative activity. With the exception of severe aplastic anemia, in which by virtue of the disease process, the marrow may be acellular, or severe combined immune dificiency in which lymphoid cells are absent, antihematopoietic activity is necessary to create space for newly transplanted cells and to eliminate abnormal stem cell clones from the marrow. While many studies have documented the antihematopoietic effects of agents in vitro, measurement of antihematopoietic effects in vivo are often difficult to interpret. This is especially true for autologous BMT where specific engraftment cannot be differentiated from recovery of non-eradicated marrow progenitors. IV. Busulfan and dimethylbusulfan

Busulfan is an effective antihematopoietic agent which may be substituted for total body irradiation (TBI) in conditioning regimens. Its antihematopoietic activity is comparable to TBI. However, busulfan lacks the immunosuppressive properties typical of radiation, so the addition of immunosuppressive agents may be required in combination with busulfan. Dimethylbusulfan is an analogue of busulfan that can be administered intravenously. It is a potent myeloablative agent, but similar to busulfan it lacks immunosuppressive properties [33]. I V-A. Phurmacology and pharmacokinetics Busulfan

Busulfan is a bifunctional alkalating agent that inhibits cell division by interaction with DNA and thiol groups on proteins [34-361. Dunn et al. postulate that busulfan preferentially inhibits cells with a low growth fraction. This may be responsible for busulfan’s specific action on pleuripotent stem cells [37]. The pharmacokinetics of busulfan have not been completely evaluated

primarily due to the lack of sensitive assay techniques. Absorption of oral busulfan appears to be complete, with a lag phase of 0.5-2 hours occurring before busulfan is detectable in plasma [38]. Following oral administration busulfan is believed to undergo both first pass metabolism and clearance by the liver [39]. Approximately 55% of busulfan is protein bound [5]. Busulfan rapidly distributes into tissue and plasma compartments including cerebrospinal fluid. Following oral administration of busulfan (1 mg/kg) cerebrospinal fluid concentrations of 559-1180 @ml have been reported. Busulfan CSF concentrations appear to be approximately equivalent to plasma concentrations in both adults and children [5]. At least 12 metabolites have been isolated but most have not been characterized. The biologic activity and antitumor activity are associated with the parent compound rather than its metabolites, although further identification and study of metabolites is necessary [38]. Metabolites are slowly eliminated in urine and approximately IO-50% of the dose is excreted within 24 hours. Peters et al. examined the pharmacokinetics of highdose busulfan (1620 mgikg) prior to BMT. Plasma steady-state levels were in the range of 2-10 ,uM and were consistent between patients, and following multiple doses within a single patient [39]. Area under the concentration time curve appears to be linearly related to dose [40]. Following oral administration both monophasic and biphasic elimination patterns have been observed. A biexponential decay with an initial half-life of 1.3-2.7 hours and a terminal half-life of 1 to 5 days was noted [40, 411. Busulfan’s long terminal half-life suggests that residual concentrations of drug may likely be present if BMT is performed soon after drug administration. However, many pharmacokinetic studies reporting a long terminal half-life do not differentiate between the active parent compound and inactive metabolites. More recent evidence suggests that the terminal half-life of the parent compound (or cytotoxic species) may be shorter (1.8-5.6 hours) following a dose of 1 mg/kg every 6 hours x 4 [5]. Busulfan steady-state concentrations of 83 l- 1480 ngiml were measured following this same dose. From this information, an estimate of 2 days is necessary following busulfan administration to allow sufficient time for the majority of busulfan to be cleared from plasma. Of importance is that the metabolism of busulfan in children may differ from that seen in adults. Peak concentrations of orally administered busulfan is lower in children than adults and the volume of distribution is twice that of older patients [41]. This may in part account for a lower rate of engraftment [41]. By utilizing a body surface area dosage for children rather than a kilogram dosage the area under the con-

246

centration curve (AUC) of busulfan approached seen in adults [41].

that

Dimethylbusulfan Dimethylbusulfan (1 ,Cdimethylsulfonoxy butane) differs from busulfan by the addition of methyl groups to the terminal carbon atoms of the busulfan molecule. The methyl groups alter the mechanism of action of the drug. Because it is more soluble than busulfan it can be administered parenterally. Since this agent was introduced in the early 1950s prior to requirements of Phase VII pharmacokinetic studies, very little is known about the pharmacokinetic behavior of dimethylbusulfan, and further studies should be done prior to the routine incorporation of this agent into BMT conditioning regimens. I V-B. Immunosuppression Busulfan Although busulfan is markedly myelosuppressive, it has virtually no immunosuppressive effects and may even act as an immunologic adjuvant in some cases [42]. Santos and Tutschka examined the effects of syngeneic and allogeneic marrow infusion following lethal doses of busulfan in rats. A lowering of peripheral lymphocyte counts was noted, but T- and B-lymphocyte areas in the lymph nodes and spleen were not altered [43]. Peters et al. examined the immunological properties of busulfan in patients with solid tumors receiving high-dose busulfan (16-20 mg/kg over 4 days) prior to BMT. There was no significant change in absolute lymphocyte and T-cell subset counts throughout therapy. However, unusual transient autoimmune disorders were observed in 3/6 patients. Severe seronegative arthritis, antiplatelet antibodies and chronic active hepatitis were also noted with this very high dose of busulfan [39]. Dimethylbusulfan Elson et al. compared the immunosuppressive effects of different members of the myleran series and found dimethylbusulfan to be similar to other myleran analogs. Only slight lympholytic activity was noted [44]. Since dimethylbusulfan is a weak immunosuppressive agent, BMT regimens incorporating this agent for nonautologous transplants may require further immunosuppressive activity. IV-C. Antihematopoietic

activity

Busulfan Busulfan is a very potent myelosuppressive agent which is capable of producing a profound marrow aplasia. Busulfan selectively inhibits hematopoiesis without

affecting lymphocytes [45]. It is considered equally as potent as irradiation in its hematopoietic stem cell ablative properties [46,47]. Mauch et al. compared the efficacy of TBI and busulfan as a preparative regimen in mice. Both TBI and busulfan were potent stem cell killers and produced a dose-dependent decrease in bone marrow colony forming units (CFU). When doses that resulted in equivalent donor marrow engraftment were compared, CFU survival was lower following TBI versus busulfan. The authors suggest that busulfan may selectively kill an earlier stem cell population than those killed by TBI [48]. Trainor et al. compared the antihematopoietic properties of busulfan and BCNU. Granulocytic progenitor cells (CFU-C) and pluripotent stem cells (CFU-S) were assayed in mice following short-course therapy. Busulfan (20 mg/kg) and BCNU (30 mg/kg) were administered at intervals of 2 weeks. Both busulfan and BCNU were similar in their inhibitory effects on CFU-C compared to controls (10 and 11% survival capacity). Busulfan was slightly more effective than BCNU as measured by a CFU-S assay (25% versus 35% of the controls [49]). The clinical efficacy of busulfan in conditioning regimens may depend on the disease being treated. Patients with acute non-lymphocytic leukemia have successfully engrafted following the use of busulfan (4 mg/kg/day x 4 days) alone; however, results are more variable in other diseases [50]. Patients with Hurler’s and Sanfilippo B disease, receiving busulfan (2 mglkgiday x 4 days) prior to BMT have required further antihematopoietic therapy in order to achieve hematopoietic engraftment [51, 521. Parkman et al. compared the antihematopoietic activity of busulfan and TBI in patients with congenital disorders prior to receiving BMT. Sustained engraftment was noted in 616 patients receiving TBI (750-900 R at 5 rad/min, day-l) and in 6/8 patients receiving busulfan (2 mg/kg orally on days -9, -8, -7 and -6). Higher doses of busulfan (3 or 4 mgikglday x 4 days) may be required to achieve complete antihematopoietic effects in congenital disorders. Lucarelli et al. achieved a 77% disease-free survival at >700 days postallogeneic BMT in 30 children with thalassemia receiving busulfan (4 mg/kg/day x 4 days) in combination with cyclophosphamide (50 mg/kg/day x 4 days) [53]. Dimethylbusulfan Pharmacologically dimethylbusulfan is estimated to be more than twice as potent as busulfan in its antihematopoietic activity and has a faster onset of action [54]. Whereas busulfan primarily affects stem cells, dimethylbusulfan appears to have direct cytotoxic effects on mature cells in addition to suppressing the stem cell population. Phase I studies using dimethylbusulfan as

241

conditioning for advanced malignancies prior to autologous BMT resulted in sustained engraftment in 9/l 1 patients with doses of 6-10 mg/kg/ x 1, indicating that adequate antihematopoietic activity may be achieved using dimethylbusulfan [55]. IV-D.

Antineoplastic

activity

Busulfan

Busulfan is routinely used in the treatment of chronic myelogenous leukemia. Its role as a conditioning agent in BMT for acute leukemia is currently under study. Clinical trials in acute non-lymphocytic leukemia comparing busulfan/cyclophosphamide and irradiation/cyclophosphamide conditioning prior to allogeneic BMT demonstrate that busulfan/cyclophosphamide may be therapeutically equivalent if not superior to cyclophosphamide and TBI. This may be due to the previously noted additive effects between cyclophosphamide and busulfan in acute leukemia [50]. Busulfan may also be used in the treatment of solid tumors. Busulfan preferentially inhibits cells with a low growth fraction which is often the case with solid tumors. High-dose busulfan may be ideal for autologous BMT of solid tumors since it provides excellent antihematopoietic activity and antineoplastic properties without the complications of immunosuppression [39]. Limited activity has been reported for busulfan in melanoma, with or without BMT [39, 561. The use of busulfan to eradicate menigeal leukemia is not well understood at this time, although busulfan readily penetrates into this sanctuary. Dimethylbusulfan

High-dose dimethylbusulfan has demonstrated antineoplastic activity in a number of solid tumors. Complete responses have been reported in patients with Ewing sarcoma, but responses are not always durable [55]. Dimethylbusulfan has more recently been evaluated as a conditioning agent prior to BMT in ALL, AML and other refractory leukemias with some success [57]. Bierman et al. report that dimethylbusulfan has produced remissions in patients with chronic granulocytic leukemia [58]. IV-E.

Toxicity

antagonist has significantly decreased the incidence of gastrointestinal symptoms during the preparative regimen which has included many agents [58]. In addition, hyperbilirubinemia (bilirubin ~5.0 mg/dl) and jaundice were noted in up to 50% of patients. Hepatic sinusoidal fibrosis has also been reported with very high doses of busulfan. Furthermore, an unexplained lupus-like syndrome and chronic active hepatitis were noted [39]. ‘Busulfan lung’ or bronchopulmonary dysplasia is a severe and often fatal side effect of busulfan which occurs with long-term therapy but has not been reported frequently in the BMT literature. Less frequent toxicities have included ‘Addison’s like’ wasting syndrome and skin hyperpigmentation 159-611. Combination therapy with cyclophosphamide does not appear to alter the toxicity profile of busulfan in most studies. Dose escalation of busulfan (8-20 mg/kg) in the presence of cyclophosphamide (50 mg/kg x 4) does not alter the maximum tolerated dose of busulfan (16 mg/kg) [39]. However, in a recent study the toxicity noted in patients receiving a preparative regimen of busulfan-cyclophasphamide was greater than in comparable patients receiving cyclophosphamide and total body irradiation [39]. This non-randomized study noted an increase in the incidence of both veno-occlusive disease and hemorrhagic cystitis in the busulfan-treated patients. Dimethylbusulfan

Toxicities associated with dimethylbusulfan include mucositis, diarrhea, vomiting, skin rashes and interstitial pneumonitis. A high incidence of severe hepatic venoocclusive disease (VOD) has been noted with this agent. Shulman et al. analyzed the incidence of VOD in leukemia patients following various BMT conditioning regimens. The risk of a leukemia patient developing VOD following BMT with dimethylbusulfan (7.5-15 mg/kg) was 43% versus 11% for regimens containing Ara-C, BCNU or TBI. Although the etiology of VOD is not well understood, it has been correlated with dose intensity of the treatment regimen and prior hepatic disease. The type of BMT did not appear to effect the prevalence of BMT conditioning regimen induced VOD (allogeneic=29/191, syngeneic= l/7, and autologous=2/6) [57].

BusulJirn

V. Carmustine

Peters et al. evaluated the dose limiting toxicities of busulfan when used as a sini’ ’ agent prior to BMT. The maximum tolerated total dose of busulfan was 16 mg/ kg. The dose limiting toxicities included diffuse mucositis, diarrhea, nausea and vomiting in 83-100% of patients. Recent experience with ondansetron, a 5-HT3

Carmustine (BCNU) is a potent antihematopoietic and antineoplastic agent. These properties make it particularly useful in conditioning regimens used to treat neoplastic disease. It is currently used in conditioning of patients with leukemia, small-cell lung cancer, brain

248

neoplasms, Hodgkin’s disease and non-Hodgkin’s phoma.

lym-

V-A. Pharmacology and pharmacokinetics BCNU is a nitrosourea derivative with alkalating activity. Direct DNA interstrand crosslinking, DNA protein crosslinks, and DNA single-strand breaks have been observed [62, 631. BCNU is a highly lipophilic agent which readily penetrates the blood brain barrier and enters cells by passive diffusion [8]. BCNU is also highly protein bound (77%). Following intravenous administration, BCNU is rapidly metabolized by hepatic microsomes and cleared from plasma [7]. BCNU decomposes in vitro into a number of alkylating and carbamoylating moieties including chlorethyl isocyanate (a highly reactive intermediate), 2-chlorethylamine hydrochloride, vinyl chloride, acetaldehyde, nitrogen, and carbon dioxide [6466]. In vivo metabolism of BCNU remains unclear; however, the delayed toxicities following BCNU administration have been associated with BCNU metabolites. Several of these metabolites are noted to be biologically active, although their contribution to antitumor, immunologic and antihematopoietic activity is unknown [66, 671. Mbidde et al. studied the pharmacokinetics of BCNU (800 mg/m’) prior to autologous BMT. Peak plasma concentrations were in the range of 11.9-23.5 pug/ml 161. The distribution half-life was 32 minutes and the elimination half-life was 4.26 hours. Due to the short half-life of the BCNU parent compound it would be anticipated that cl.5 ,@ml should be present in plasma after 20 hours (4 half-lifes). However, BCNU was still detectable 24 hours after administration, which suggests a more prolonged half-life. Variability in pharmacokinetics between patients may relate to the percent body fat and serum lipid concentration [68]. A late unexpected myelosuppression was noted in 4 patients (receiving BMT ~20 hours after BCNU administration) and was believed to be associated with residual cytotoxic drug effects on marrow autografts [6]. Due to the variability in pharmacokinetics of BCNU a ‘drug free’ period of 2 days may be required to assure clearance of residual drug. The presence of active metabolites further complicates the scheduling of this agent. Pharmacokinetic monitoring of individual patients, especially just prior to BMT would be of benefit. However, further assay development and characterization of metabolites is necessary before routine monitoring can be accomplished. V-B. Immunosuppression BCNU

has demonstrated

significant

immunosup-

pressive activity in mice. Primary immune responses were examined by administering BCNU (30 mg/kg), 2 days prior to an intraperitoneal injection of sheep red blood cells (SRBCs). Antibody plaque forming units (PFUs) were then measured in mouse spleens 4 days after injection. BCNU suppressed both IgM and IgG PFUs. BCNU was also found to reduce the responsiveness of splenocytes to mitogens (T-cell and B-cell stimulants). Suppression of splenocyte response to mitogens was 90% and lasted for up to 9 days. The authors suggest that BCNU inhibits both the primary response and the development of memory cells. In addition, BCNU also inhibited immediate hypersensitivity reactions of mice to SRBCs. However, delayed hypersensitivity reactions to SRBCs might be potentiated by BCNU [69]. Unfortunately, little clinical evidence is available to confirm these reports. V-C. Antihematopoietic activity BCNU is a very effective antihematopoietic agent which causes direct suppression of hematopoietic progenitor cells [70, 711. Xu et al. examined the in vivo effects of BCNU (20, 25, 30 mg/kg) on spleen colony forming cells (CFU-S), granulocyte-macrophage colony forming cells (CFU-GM), and fibroblastoid colonyforming cells (CFU-F) in mice. Following BCNU (30 mg/kg) administration, CFU-S and CFU-GM were inhibited by 64%. The stromal cell population (CFU-F) was also effected with 67-83% of controls remaining at 150-250 days post BCNU administration [72]. V-D. Antineoplastic activity BCNU has antineoplastic activity in a variety of solid tumors and hematologic malignancies. BCNU is often used in primary and metastatic brain tumors including astrocytomas and malignant gliomas [73-751. Phillips et al. examined the use of BCNU monotherapy (10501350 mg/m’) with autologous BMT in 36 malignant glioma patients, with 12/27 patients with progressive disease ‘responding to therapy. A prolonged progression-free survival was noted in these patients; however, fatal treatment related toxicities occurred in 16% of patients [76]. BCNU is also active in Hodgkin’s and non-Hodgkin’s lymphoma. It is commonly administered in combination conditioning regimens containing cyclophosphamide and etoposide, resulting in complete and partical responses in up to 50% of patients [77-801. BCNU has also been examined in patients with acute leukemia, melanoma and breast cancer with less conclusive results.

249

V-E. Toxicity

VI. Melphalan

Pulmonary fibrosis and interstitial pneumonitis are frequently seen following conditioning with high-dose BCNU. The incidence of BCNU induced pulmonary fibrosis is dose-related, with high doses (1050-1200 mg/ m2) producing pulmonary fibrosis in approximately 10% of patients [76]. Severe pulmonary fibrosis may occur in patients receiving total doses of BCNU > 1400 mglm’. Combination therapy with other cytotoxic agents may increase the risk of BCNU induced pulmonary toxicity. A combination of high-dose cyclophosphamide, etoposide and BCNU demonstrates a dose-related increase in BCNU induced pulmonary toxicity. Patients receiving BCNU (450 mg/m’) had an incidence of l/7 patients with interstitial pneumonitis compared to patients receiving BCNU (600 mg/m2) with an incidence of 5/18 [80]. BCNU pulmonary toxicity may also be related to the scheduling of the drug. Phillips et al. report 5/42 deaths due to interstitial pneumonitis following bolus doses of 600 mg/m2, versus l/l8 deaths when the same dose was administered over 4 days in divided doses [79]. Severe hepatotoxicity has also been noted following high-dose BCNU. The incidence may be as high as 26%. In one study, venoocclusive disease occurred in 4110 patients with hepatitis occurring in l/l0 patients receiving BCNU (600 mg/m’ in 8 divided doses every 12 hours). A correlation between hepatotoxicity and pharmocokinetics was noted, with higher BCNU area under the concentration curve (AUC) in patients experiencing hepatotoxicity. Administration of BCNU (600 mg/m2) in divided doses may help to reduce the incidence of venoocclusive disease [80]. Hepatotoxicity may also be increased in patients administered other hepatotoxic agents. Administration of low doses of BCNU ( 150 mg/ m2 x 3) in combination with cyclophosphamide (120 mg/kg) and TBI (1000 rads) produced acute liver failure and mortality in 3/6 leukemia patients receiving allogeneic BMT [8 I]. Dose reduction in hepatic disease may be required; however, dosing guidelines remain to be established [82]. BCNU induced gastrointestinal side effects include nausea, vomiting, diarrhea, esophagitis. anorexia and dysphagia. Fatal encephalomyelopathy at 1050 mg/m2 and late unexplained neurologic deterioration have been reported [76]. Other side effects may include progressive azotemia, dizziness and flushing of the skin, hypotension, tachycardia and peripheral vasodilation. Cardiovascular side effects may be associated with the ethanol diluent. The amount of ethanol used to dissolve BCNU should be kept at a minimum [83]. Administration of BCNU twice daily for 8 doses has also reduced these side effects [82].

Melphalan has primarily been used in combination BMT conditioning regimens for patients with solid tumors (neuroblastoma, melanoma, sarcoma and breast cancer). It is an excellent antihematopoietic and antineoplastic agent; however, its use has been associated with severe toxicities which continue to limit its use. VI-A. Pharmacology and pharmacokinetics Melphalan is a bifunctional alkalating agent which inhibits DNA replication and transcription of RNA. Following intravenous administration, melphalan distributes into a volume equivalent to total body water and quickly binds to plasma proteins. It is >80% protein bound 4 hours after intravenous administration [84]. First-order elimination from plasma occurs following first-order hydrolysis, with monohydroxy and dihydroxy metabolites appearing in urine. Following highdose melphalan (140-180 mgm’), the elimination halflife is 40-50 minutes [16]. Greater than 90% of melphalan is cleared from plasma after 4 hours. The halflife of the hydroxylated metabolites is 2-3 times that of melphalan. These metabolites have been reported to be only weakly cytotoxic; however, they may have significant antihematopoietic activity [85]. Peak concentrations of parent drug after a 0.6 mg/kg dose are approximately 280 ng/ml. Combination BMT regimens with cisplatinum, cyclophosphamide and escalating doses of melphalan report a shorter half-life of 20-30 minutes, with a linear increase in melphalan’s AUC with increasing doses. Patients with renal failure have reduced clearance of melphalan and a prolonged half-life [86]. Melphalan distributes into the cerebral spinal fluid at about 10% of the plasma concentration following high-dose therapy [87]. VI-B. Immunosuppressive activit+y Melphalan is transiently immunosuppressive in mouse studies. Sagi et al. examined the long-term effects of melphalan (250400 ,ug, i.m., day 14 and 24) on immune parameters of mice with and without prior plasmacytoma. Melphalan was reported to reduce Tcell function up to 120 days after drug administration as measured by splenic cell response to alloantigens. T-cell numbers measured by monoclonal Anti Thy 1.2 antibodies were reduced up to 60 days following melphalan. In addition, NK activity of splenic cells was markedly suppressed initially after melphalan with recovery to normal after day 80-90. B-lymphocyte numbers were initially reduced as measured by the number

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of splenic surface immunoglobulin (SIg) positive cells, with recovery occurring by day 120 [88]. VI-C.

Antihematopoietic

activity

Melphalan may cause a permanent reduction in stem cell growth and autoreconstitive capacity [72, 891. Following in vitro melphalan exposure (6.6 PM x 1 hour) CFU-C are inhibited by 80%. However, long-term exposure ( 1O-4 M x 8 hours) may be required to achieve complete inhibition [go]. Worthington-White examined the effects of single versus multiple exposures to melphalan. Repeat exposure of melphalan ( 10m4M for 1 hour x 8) versus single exposure (10m4M; 8 hours x 1) decreased plating efficiency from 60% to 5%. The authors conclude that melphalan’s antihematopoietic activity was greatest with prolonged and repeated exposure. In addition, evidence was also presented suggesting that the metabolites of melphalan may have significant antihematopoietic activity. Future research is necessary to determine the antihematopoietic activity of these metabolites. VI-D.

Antitumor

activity

High-dose melphalan (180 mg/m’ over 30 min) prior to BMT has been used in advanced breast cancer and metastatic colon cancer. Overall response rates in metastatic colon cancer have been reported up to 45%. Leff et al. report complete response in 3/15 and partial responses in 6/l 5 colon cancer patients [9 11.Peters et al. evaluated the use of melphalan (40-150 mg/m’/d, day-4) in combination with cisplatin (55-60 mg/m’/d x 3 days), and cyclophosphamide (1875 mg/m2/d x 3) prior to autologous BMT in patients with advanced breast cancer, melanoma and sarcoma. They report frequent, rapid responses with objective clinical response in 78% of patients. Responses were seen in all breast and sarcoma patients and in 6/l 1 patients with melanoma. However, the duration of response and survival times were not durable [86]. Melphalan (140 mg/m’) has also been examined as a conditioning agent prior to autologous BMT for patients with advanced neuroblastoma. In one study where melphalan/BMT was administered as part of a multimodality treatment with vincristine, adriamytin, cyclophosphamide and radiation, 318 responses were noted which lasted 6, 11 and 16 months after BMT [79]. In another study, high-dose melphalan (140-180 mg/m2) as single-agent conditioning, produced no significant antitumor responses in patients with measurable disease, although further consolidation therapy with melphalan was associated with improved survival, 5/8 patients alive and free of disease at 29-54 months [93].

VI-E.

Toxicity

Nausea, vomiting, diarrhea, anorexia and stomatitis are common side effects of melphalan. Mild transient liver toxicity has been reported in 20% of patients. Tumor necrosis with subsequent perforation has been reported in several patients receiving high dose melphalan [91, 941. Dermatologic reactions including urticaria, pruritus and anaphylaxis have occurred in patients even at low doses. Renal impairment may reduce the clearance of melphalan and should be monitored closely in order to reduce the dose if necessary.

VII. Radiation

Total body irradiation is a commonly used and reliable hematopoietic ablative and immunosuppressive agent. It may be incorporated as either a single or fractionated dose in the design of conditioning regimens. Additional electron beam and rib ‘boosting’ may be used to provide further antineoplastic activity. In circumstances where only immunosuppressive effects are needed (e.g., aplastic anemia), radiation may be given as ‘total lymphoid irradiation’ (TLI) or thoraco-abdominal radiation.

VII-A.

Total body irradiation

(TBI)

The mechanism of cell inhibition induced by radiation has been examined by a number of investigators. Following lethal doses of irradiation, most cells die by a process which is mitotically linked. However, some cells including unstimulated peripheral lymphocytes may die in interphase. Following irradiation there is a fall in circulating lymphocytes followed by a delayed inhibition of erythropoiesis and thrombocytopenia [95]. A variety of radiation rates, qualities, doses and dose rates have been reported. Total doses used can be in the range of 800-1500 rad, given as a single dose or in divided fractions at a low-dose rate of 5-20 rad/min. While dose-limiting toxicities to non-hematopoietic organs, particularly the lung, have prevented further dose escalation of radiation, variations in dosing and combination therapy with other agents have been evaluated. VII-B.

Immunosuppression

TBI is an effective immunosuppressive agent; however, in order to achieve complete immunosuppression in the case of increased genetic disparity between donor

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and recipient, a second immunosuppressive agent may be required [96-981. The amount of post-radiation immune suppression is dependent on the extent and location of the radiation field, and the irradiation dose rate and fraction [99, 1001. TBI is a potent inhibitor of lymphocytes. Lymphocytes appear to be the most radiosensitive of all tissues. The immunocytotoxic effects of TBI are most potent for B cells; however, T-cells are also affected. Following TBI (1 Gy) a 25% reduction in circulating lymphocytes can be seen 4 hours after irradiation [loll. The cytotoxic effects of TBI on T-cells is dependent on the T-cell subset and prior activation. Studies demonstrate that unprimed T-helper and T-suppressor cells are sensitive to irradiation while primed T-helper cells appear to be relatively resistant [ 1021. Cell-mediated responses are therefore less sensitive to irradiation than the production of antibodies. Secondary (‘memory’) immune responses mediated by T-cells are the least sensitive. From a clinical point of view low-dose TBI, thoraco-abdominal irradiation and total lymphoid irradiation have all been successfully utilized to prevent graft rejection. VII-C. Antihematopoietic activity Cell survival following radiation is a constant exponential function of the dose administered. A constant proportion of cells is killed rather than a constant number. The dose of radiation which is required to achieve a 37% (e-‘) survival fraction is defined as the D,. In most cultured mammalian cells, including stem cells, the survival curve, plotting log of the surviving fraction versus dose of radiation, is linear at doses of radiation greater than 3 Gy. The terminal slope is described by the D,. At low doses (~3 Gy) a non-linear curve (or ‘shoulder’) exists where there is reduced efficacy in cell killing. The shoulder may be described by D, in the equation e”=D,l D,. Where D, (quasi-threshold dose) may be considered a measurement of the sensitivity of particular cells to the sublethal damage repair of radiation and n is the extrapolation number at the initial shoulder region. Bone marrow stem cells appear to have a very low ns (0.6) compared to other cells (skin, intestine, lung). When a dose of radiation is divided into two fractions given a few hours apart there is a return of the ‘shoulder’ region, suggesting that split dosing may be less effective than a single dose for most tissues other than hematopoietic tissue. Dose fractionation may therefore protect most cells against radiation induced cytotoxicity, while, bone marrow stem cells will remain sensitive to fractionated doses due to their low D, [103]. This strategy can then be employed to improve the therapeutic ratio of total body irradiation.

VII-D. Antineoplastic activity Doses of irradiation required to achieve local antitumor effects in vivo (>15 Gy) are higher than those used in TBI (10 Gy). Therefore, in order to achieve adequate antitumor effects from radiation to sites of extensive disease, additional ‘boost’ doses may be given locally to the bulk disease. This may be particularly beneficial for patients with solid tumors including lymphomas and Hodgkin’s disease. ‘Boost’ doses of radiation allow for large doses of radiation to be delivered to a local area, allowing for enhanced antitumor effects while keeping systemic radiation exposure to a minumum. Localized ‘boost’ radiation has been examined in patients with diffuse histiocytic lymphoma receiving TBI and cyclophosphamide as a conditioning therapy prior to autologous BMT. Patients receiving conditioning with hyperfractionated TBI (1320 to 1375 cGy in 11 fractions over 4 days), cyclophosphamide (60 mg/kg/d) and ‘boost’ radiation to areas of residual disease had enhanced antitumor activity compared to cytotoxic treatment alone [104]. Although ‘boost’ radiation may reduce the risk of systemic toxicities, toxicity to any given organ may be greater due to the additive effects of TBI and boost doses to that organ. VII-E. Radiation-induced toxicity The severity and frequency of irradiation induced toxicities are dependent on the dose, dose fraction, dose rate, and the use of other cytotoxic agents. Radiation induced toxicities occur in a variety of tissues; however, cells of the lung, thymus and gastrointestinal tract are more sensitive than other tissues. Pulmonary toxicities including pneumonitis are frequently seen in patients receiving TBI prior to BMT. Pneumonitis may be a result of depletion of Type 2 alveolar cells and endothelial cells resulting in an osmotic imbalance in the lung. The incidence of pneumonitis is dose-related, doses of 8.3 Gy produce pneumonitis in 5% of patients while doses of 9.5 Gy produce a 50% incidence [105]. While the incidence of pneumonitis in leukemic BMT recipients has been reported as high as 40% it is likely an overestimation of pneumonitis caused by radiation alone since cytomegalovirus and other infections produce a similar clinical picture [106]. The overall incidence may be less in autologous and syngeneic BMT in part due to the lesser post-transplant immunosuppression given to by these patients; however, these patients also typically do not receive post-transplant prophylaxis with methotrexate for GVHD which may also contribute to pulmonary toxicity. There is also a lower incidence of CMV-related interstitial pneumonia in pa-

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tients receiving syngeneic versus allogeneic BMT [107]. Combination immunosuppression therapy and GVHD may also be contributing factors to the development of irradiation induced pneumonitis [ 1081. It should also be noted that many other agents such as busulfan also cause a similar frequency of post-BMT pneumonitis. Other side-effects include thyroid dysfunction, growth retardation in children, nephro- and hepatic toxicity and cardiac failure [109, 1lo]. Cataract formation is frequently seen in patients receiving single-dose TBI (80% with 10 Gy, within 6 years); however, with fractionated doses (6 x 2 Gy or 7 x 2.25 Gy) it is reduced to 18% [l 111.Cardiac complications from irradiation may be immediate (days), short-term (months) or late (many years) [112]. Cardiac toxicities may manifest as acute pericarditis, fibrotic thickening, pericardial effusion, constrictive pericarditis, and restrictive cardiomyopathy. The incidence of cardiotoxicity may increase when TBI is combined with high-dose cyclophosphamide or in patients who previously received anthracyclines. Cardiotoxicity is generally associated with large doses of irradiation rather than those used in TBI [1131171.

VIII. ‘Nonablative’

antineoplastic

agents

The use of antineoplastic agents with minimal activity against pluripotent hematopoeitic stem cells has increased in conditioning regimens. The increase is primarily due to the improved response rates noted with dose escalation of antineoplastic agents. Typically, dose escalation of antineoplastics is limited by bone marrow suppression. The use of BMT rescue has allowed for further dose intensive therapies although in many cases the absolute need for bone marrow rescue is unclear. Antineoplastic agents used in BMT regimens should be specific for the type of neoplasm involved and should preferably have some degree of selectivity for neoplastic stem cell clones in hematologic malignancies. Agents must also have adequate penetration to the site of involvement. VIII-A.

Cytosine arabinoside (Ara-C)

Ara-C is primarily incorporated in BMT conditioning regimens for its antineoplastic activity. High-dose Ara-C has demonstrated antineoplastic activity in patients refractory to conventional doses of Ara-C [118]. Ara-C may also be used in combination with cyclophosphamide and/or TBI as a preparative regimen for acute and chronic leukemias [ 1191.

Pharmacology and pharmacokinetics

Cytosine arabinoside is an inactive arabinose nucleoside derivative that undergoes intracellular metabolism to an active S-triphosphate (Ara-CTP) metabolite. Following intracellular activation, Ara-CTP inhibits DNA synthesis by competitive inhibition of DNA polymerases [ 120, 1211. A number of experimental models show that Ara-C activity is schedule-dependent [122]. In vitro experiments demonstrate that high concentrations of Ara-C (1 pug/ml) for brief periods are lethal to cells, while concentrations of ~0.1 ,&ml may inhibit DNA synthesis but are only cytostatic [123]. Following administration Ara-C is inactivated by deamination in the liver, kidneys and RBCs to uracil arabinoside [2, 1241. Ara-C is rapidly cleared from plasma with ~90% of the parent compound remaining in plasma 1 hour after infusion. However, its active intracellular metabolite Ara-CTP is retained intracellularly. Up to 1342% of Ara-CTP is retained at 4 hours following Ara-C infusions (100 mg/m’/ day x 10). Longer retention of this active metabolite has also been correlated with improved clinical response in ANLL patients [ 1251. In order to design a rational BMT conditioning regimen with high-dose Ara-C further evaluation of the active Ara-CTP metabolite is warranted. The pharmacokinetics of the parent drug vary between patients. Following intravenous administration of Ara-C (l-3 g/m’), plasma concentrations are dose proportional. Initial and terminal half-lives (7.6.-15.6 min and 2.3-2.6 hours, respectively) are approximately the same at low dose (100 mg/m’) and high-dose (3 g/m’). Accumulation of Ara-C does not appear to occur even after successive doses, suggesting no saturation of deaminase enzymes occurring within the dosing range of l-3 g/m’. Ara-C penetrates the blood brain barrier and rapidly equilibrates between the plasma and cerebrospinal fluid. Slevin et al. report that CSF concentrations of Ara-C rise linearly with increasing doses (1-3 g/m*). Following 3 hour infusions CSF concentrations are 622% of plasma Ara-C concentrations and ranged from 0.35-l .07 pug/ml. CSF concentrations decline with a half-life of 2 hours [l]. Zmmunosuppression

Ara-C has been demonstrated to have mild in vivo immunosuppressive effects. Gassman et al. compared the immunosuppressive properties of Ara-C with cyclophosphamide in a busulfan-treated rat model prior to BMT. Rats were given Ara-C (75 mg/kg) or cyclophosphamide (60 mg/kg) prior to BMT. Immunosuppression 100 days post-BMT was measured by transplantation of allogeneic skin grafts to indicate persistence or rejection of the transplanted marrow. No rejec-

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tion was noted in cyclophosphamide treated rats, while a rejection rate of 75% was noted in Ara-C-treated rats [ 1261. Clinical studies replacing cyclophosphamide/TBI with Ara-CTBI have resulted in engraftment and some immunosuppression in the Ara-C regimen.

variety of other side effects have been reported with this combination including mucositis, nausea and vomiting, diarrhea, skin rash, photophobia, conjunctivitis, necrotizing colitis or ileus, elevation in liver enzymes, and SIADH [129, 1341.

Antihematopoietic

VIII-B.

activit?

The antihematopoietic effects of Ara-C are time and concentration dependent. Muus et al. report that Ara-C cytotoxicity to clonogenic cells is related to the proliferative and cycling state of the progenitor cells and exposure time [ 1271. Continuous Ara-C given a prolonged period may be necessary to adeablative Leach et examined effects of Ara-C on mouse When of Ara-C mglkgldose q 3 hours 24 hours) given 3 days the on the hematopoietic system were transitory. Recovery was rapid, with complete recovery in 4872 hours [I 281. However, with continuous uninterrupted doses (up to 360 mg/kg over 72 hours), a progressive disappearance of granulocytic stem cells was noted. An irreversible aplasia occurred with only rare cells belonging to the lymphocytic series and a few scattered histocytes remaining. Histologic changes in the spleen and lymph nodes were also noted. There was an absence of mitotic activity in the germinal centers and splenic pulp and the splenic pulp showed an absence of hematopoietic cells and megakaryocytes. Antineoplastic

activity

Ara-C is selective for actively dividing cells and rapidly proliferating tumors. Concentrations as high as 2500 &ml have been reported to be ineffective at killing non-dividing cells [122]. Ara-C has been shown to be particularly active in acute leukemias, and has shown some activity in lymphomas. Coccia et al. studied highdose Ara-C (3000 mgim’ given in 1 hour every 12 hours x 6 days) followed by TBI (six doses of 200 cGy twice daily for 3 days) prior to allogeneic BMT in 20 children with acute lymphoblastic leukemia. The regimen had a substantial antileukemic effect with 1l/l 8 ALL patients in second remission achieving continuous complete remission 12-79 months after BMT [129]. Toxicity

Adverse effects associated with high-dose Ara-C in BMT conditioning regimens include severe CNS, pulmonary and gastrointestinal toxicity. Cerebral and cerebellar dysfunction may occur more frequently in older patients, and in patients receiving large doses (>3 g/m’) [13&l 321. Ara-C may be associated with a syndrome of sudden respiratory distress, progressing to pulmonary edema and cardiomegaly which may be fatal [133]. A

Thiotepa

Thiotepa has antineoplastic activity in a variety of solid tumors. It is currently being examined as an active antineoplastic agent in several conditioning regimens. The antihematopoietic and immunosuppressive effects of this agent remain to be studied at the doses used in BMT. Pharmacology

and pharmacokinetics

Thiotepa is an alkalating agent that inhibits DNA replication and transcription of RNA. Current evidence suggests that thiotepa may act as a prodrug requiring enzymatic activation [135]. Thiotepa is metabolized in the liver to tepa and other alkalating metabolites [25, 1361. Plasma concentrations of tepa remain equal to or exceed that of thiotepa [136]. The steady state volume of distribution for thiotepa is 0.7 1 liter/kg which implies distribution throughout total body water. Thiotepa also distributes into the CSF. CSF levels measured 15 minutes after administration of thiotepa were equivalent to plasma concentrations [27]. Binding of thiotepa to serum albumin and lipoproteins is small (10%) [28]. Following a dose of 0.9 mg/kg peak plasma levels of drug reach approximately 1 pug/ml. Although the parent compound is rapidly cleared from plasma, the cytotoxic tepa metabolite is cleared much more slowly. Total exposure to tepa is 2-fold greater than thiotepa [27]. In a phase I dosing escalation study of thiotepa (135-1215 mg/m’ iv. over 3 days), prior to autologous BMT, a biexponential decay curve was noted with a thiotepa distribution half-life of 0.11 hours and an elimination half-life of 2.6 hours [24]. There is substantial interpatient variability in systemic clearance of thiotepa which may significantly effect individual patient exposure (AUC) [137]. Saturation and induction of thiotepa clearance was observed when combined with cyclophosphamide [ 1381. Due to the presence of active metabolites and interpatient variability of this agent, pharmacokinetic monitoring of the parent drug and metabolites prior to BMT may improve the efficacy of this agent. Immunosuppression

In vitro studies by Mourelatos et al. report that thiotepa is cytotoxic to peripheral human lymphocytes at concentrations of 60 ng/ml as measured by sister chro-

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matid exchange methods (SCE). The effects on lymphocytes appear concentration dependent, with larger concentrations causing increased cytogenetic damage [ 139, 1401. Low-dose thiotepa (30 mg x 2 days then every 14 days for 10 weeks) reduces natural killer cell (NK) activity, and leucocyte subpopulations in human peripheral blood. However, the effects of thiotepa on myelopoesis and lymphopoiesis need further study [141].

developed in patients receiving doses of 7 mg/kg x 3 [145]. Central nervous system toxicity has been reported in patients receiving doses of 1000 mg/m* [146]. Other side effects include nausea, vomiting, anorexia, diarrhea, alopecia, headache, dizziness and hyperuricemia and dermatologic toxicities.

Antihematopoietic activity Little information exists on the antihematopoietic effects of thiotepa. In vitro assays examining the antihematopoietic activity of thiotepa are difficult to interpret since the metabolites are considered the primary active species. Teicher et al. examined the in-vivo antihematopoietic effects of thiotepa on mouse CFU-GM. Thiotepa (SO-100 PM) produced >90% inhibition of mouse CFU-GM. Tanum et al. report that sublethal concentrations of thiotepa (12 mg/kg) administerd to rats, reduced the total megakaryocyte numbers in the bone marrow. Thiotepa may act by decreasing or blocking the influx of progenitor cells into the megakaryocyte department [ 1421. Further characterization and study of the active metabolites of thiotepa are necessary prior to establishing the antihematopoietic nature of this compound.

Etoposide is almost exclusively marrow toxic making it an ideal agent for use in combination with BMT. Increasing doses of VP-16 lead to severe myelosuppression in humans with only minor extramedullary toxicities [147]. Teniposide is currently being explored as a BMT preparative agent in patients with acute lymphocytic leukemia [148]. Its efficacy as an antihematopoietic, immunosuppressive and antineoplastic agent in BMT regimens requires further evaluation.

Antineoplastic activity Thiotepa has demonstrated antineoplastic activity in the treatment of refractory malignancies including breast, melanoma, ovarian, non-small-cell lung and colon cancers [24, 1431. High-dose single agent thiotepa has produced objective responses in 6125 patients receiving autologous BMT in a variety of solid tumors (melanoma, adenocarcinoma, non-small-cell carcinoma of the lung, and colon cancer). Dose escalation of thiotepa does not appear to correspond to more frequent or more durable responses [24]. Phase I/II studies using high-dose thiotepa (6c52.5 mg/m2/day x 3 days), followed by autologous BMT for metastatic melanoma, noted complete responses in 4/71 patients and partial responses in 25/71 patients. The duration of these responses was 3 months (range l-31 months) with 10% of responses lasting greater than 1 year [ 1441. In combination conditioning regimens with cyclophosphamide for advanced solid tumors, overall response rates of up to 70% have been reported [145]. Toxicity Phase I studies using escalating doses of thiotepa (135-1215 mg/m2 i.v. over 3 days) prior to autologous BMT, report mucositis and CNS toxicity as dose-limiting. Severe life-threatening mucositis and enteritis have

VIII-C. Etoposide (VP-16) and teniposide (VM-26)

Pharmacology and pharmacokinetics VP-16. Etoposide (VP-16) is a semisynthetic epipodophyllotoxin derivative which induces singleand double-stranded DNA breaks [149, 1501. Following exposure to VP- 16, cells enter G-2 phase and complete DNA synthesis, but they are unable to divide [151-1531. Following intravenous administration VP- 16 rapidly distributes throughout the body into a volume approximately equivalent to total body water. VP-16 also distributes into CSF where concentrations of up to 0.54 ,@ml have been measured following high-dose therapy (0.9-2.5 g/m’) [154]. It is highly protein bound and is primarily excreted in feces. The major metabolic pathway for VP-l 6 is hydroxylation, however, a cis-picro lactone isomer, and a O-demethylation product have also been isolated [15, 155, 1561. O-Demethylation results in a reactive 3’,4’-dehydroxy metabolite (DHVP16). Reactive intermediates may bind irreversibly to hepatic microsomal proteins [I 51. It must be remembered particularly in the complexities of BMT that drugs which induce cytochrome P450 may also enhance the metabolism of VP- 16. The pharmacokinetics of VP-l 6 in BMT conditioning regimens has been demonstrated to be highly variable. Half-lifes of 3-15 hours have been reported for this agent. Blume et al. examined the pharmacokinetics of VP-16 in two patients receiving 30 mg/kg (4 hour infusion) prior to BMT. Peak plasma concentrations were 120 pug/ml at the cessation of infusion, with a terminal half-life of 7.5 hours. Approximately 50 hours after infusion VP-l 6 plasma concentrations were no longer detectable [ 131. In patients receiving VP-l 6 (2-3.5 g/m’) administered as a twice daily infusion (over l-2 hours for 3 days) a much slower elimination was noted. A

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triphasic elimination curve was noted with detectable plasma concentrations (1 O-250 ng/ml) remaining 168 hours from the start of infusion [14]. Due to the slow elimination of VP-16 and the presence of active metabolites, a drug-free period of at least 2 days may be required prior to BMT. This variability in pharmacokinetics may be dependent on a number of factors. Reduced clearance of VP-16 has been associated with elevation in serum creatinine and alkaline phosphatase, prior cisplatin therapy, obesity and age [I 57, 1581. Since VP-16 is also highly protein bound, other agents incorporated into BMT conditioning regimens which are also highly protein bound may displace VP- 16 and alter the active unbound fraction of drug. VM-26. VM-26 is an epipodophyllotoxin antineoplastic agent which acts by inhibiting microtubules. At low concentrations (1 mg/l), VM-26 irreversibly blocks cell cycle progression in late S and G2 phases. Higher concentrations produce a cytolytic effect which is nonspecific [23, 1591. Several metabolites of VM-26 have been identified, including an epiaglycone, picrolactone and hydroxyacid derivative. The epiaglycone metabolite has been noted to be a potent cytotoxic agent, although is has not been detected in human plasma [155]. The other metabolites are not considered active at the plasma concentrations measured after administration of low-dose VM-26. However, little is known about these metabolites at the higher doses used in BMT conditioning regimens. VM-26 distributes rapidly throughout the body and achieves high CSF concentrations (27% plasma concentration). VM-26 is highly bound to plasma proteins (>90%), and is eliminated more slowly than VP-16. Elimination is triphasic with a terminal half-life of 3-l 5 hours [ 1601. A terminal half-life of 10.3 hours was noted following continuous infusions of VM26 (300-750 mg/m*) in pediatric patients with leukemia. Response to therapy was associated with a decreased total body clearance, higher mean steady-state levels and long elimination half-lives. However, higher mean steady- state levels were also associated with more toxicity [22]. The pharmacokinetics of high-dose VM-26 in BMT regimens remain to be evaluated. Immunosuppression

VP-16. VP-16 is a weak immunosuppressive agent and should be supplemented with cyclophosphamide or another immunosuppressive agent in BMT conditioning regimens requiring immunosuppressive activity. Gassman et al. compared the efficacy of increasing doses of VP-16 (30-60 mg/kg x 1) and cyclophosphamide (60 mg/kg) in allowing engraftment of allogeneic bone marrow in rats following ablative doses of busul-

fan (35 mgkg). Rats received an allogeneic skin graft loo-days post-BMT to ascertain if rejection of the transplanted marrow occurred. Successful engraftment was noted in all animals receiving cyclophosphamide, while graft rejection rates of 58% were noted for VP-16. They caution against using VP-16 in place of cyclophosphamide in BMTs that have an increased risk of graft injection [126]. Little is known about the immunosuppresI/M-26. sive potential of VM-26 at the doses used in BMT. Antihematopoietic

activity

VP-16. VP-16 is extensively marrow toxic and appears to effect pluripotent committed precursors and differentiated mature cells [ 1611. Both actively dividing and resting stem cells are inhibited by VP-16. In addition, tumor clonogeneic cells may be more sensitive to VP-16 than normal committed (CFU-C) and pluripotent (CFU-S) stem cells [162]. Following in vitro exposure of VP-16 (5 pmol/l x 1 hour), 50% of neoplastic cells (SK-DHL, promyelocytic leukemia, lymphoblastic leukemia) were inhibited, while much higher concentrations (25530 pmolil) were required to inhibit normal stem cells (CFU-Mix, BFU-E, CFU-GM). Marrow stromal progenitors (CFU-F) were even less sensitive to VP- 16 (50% inhibition at 27 1 pmolil) [ 1631. VM-26 is a potent antihematopoietic agent VM-26. which may also be selective for hematopoietic neoplastic cells. Vietti et al. examined the in vivo effects of VM-26 on hematopoietic stem cells. Following administration of VM-26 (0.02,O. 15 mgmouse) hematopoietic stem cells declined rapidly and recovered slowly (20 hours). Administration of 1.2 mg/mouse resulted in a 50% inhibition of normal CFUs. In addition, leukemic cells appeared to be more sensitive to VM-26 than the normal CFUs. Administration of VM-26 as a 24 hour infusion had a better antileukemic effect than the same total dose given as a single injection [164]. Antineoplastic activity VP-16. High-dose VP- 16 has antineoplastic

activity in a number of solid tumors including small-cell carcinoma of the lung, germ cell carcinoma, leukemia and lymphoma [165]. In a phase-I dose escalation study, VP- 16 ( 1500-2700 mg/m’; given as 900 mg/m2/day x 3) was administered to 6 patients with germ cell carcinoma prior to autologous BMT. There was 1 complete response and 4 partial responses. Three of the patients showing response had previously failed standard dose VP-l 6, suggesting a dose response relationship [166]. In phase I/II studies combining high-dose VP-16 (25-60

256

mg/kg) with TBI (1320 cGy: 11 fractions of 120 cGy between days -7 and -4) in 39 patients with advanced hematologic malignancies (ANLL and ALL) prior to allogeneic BMT, there was a 43% complete response rate in patients with acute leukemia not in first remission. However, relapse and survival rates did not correlate with dose escalation of VP-16 [13]. VM-26. VM-26 has a spectrum of activity similar to VP-16 and is active in Hodgkin’s disease, nonHodgkin’s lymphoma and solid tumors including those involving the CNS [167, 1681. VM-26 has also been examined for use in conditioning regimens for acute lymphocytic leukemia patients [ 1691.VM-26 (l&l 5 mg/ kg over 2 hours) with TBI (1000-1320 cGy) prior to allogeneic BMT, produced a sustained remission in 2/l 0 patients at 1547 and 1988 days post BMT. However, 4/10 patients died of recurrent leukemia and 3/10 died of sepsis early after BMT [170].

Toxicity VP-16. Phase I toxicity studies of high-dose VP-16 prior to autologous BMT report myelosuppression and mucositis as the dose limiting toxicities. Mucositis limited the maximum tolerated dose to 2400 mg/m*. Doses of 2700 mg/m2 were associated with severe and potentially fatal oropharyngeal mucositis. No other severe toxicities were noted at these doses. Elevated liver function tests, nausea, vomiting, and rashes have been noted in other studies with high dose VP-l 6 [ 1661.Other hepatotoxic agents may increase the incidence and severity of VP-16-induced hepatotoxicity. Following dose escalation of VP-16 combined with TBI prior to allogeneic BMT, mucositis remained dose-limiting. However, severe jaundice and liver failure (grade 4) was seen in two patients receiving VP-16 (70 mg/kg) [ 131. Less severe side effects have included acute episodes of fever and chills associated with infusion, and a reversible metabolic acidosis in 43% of patients. Metabolic acidosis and transient confusion were reported to be related to the poly(ethylene glycol), ethanol solvent required as the diluent. Hypotension has been reported with rapid infusions of VP-16. V&f-26. Little information exists on the dose-limiting side effects of VM-26 as a single agent in conditioning regimens. However, phase I dose escalation studies demonstrate that similar to VP-16, mucositis is doselimiting. Moderate to severe mucositis was reported in S/12 patients following VM-26 (30&750 mg/m2/course). Acute tumor iysis, pruritus, tachycardia, bronchospasm and hypotension have also been associated with VM-26

[22]. Rapid infusions have been related to hypotension and hypersensitivity reactions (including anaphylaxis) the latter reported with both VM-26 and VP-l 6 [171]. IX. Immunosuppressive

agents

Immunosuppressive activity is the third major property of agents incorporated into BMT conditioning regimens. Immunosuppression may be necessary to guard against acute graft rejection by the host’s immune system and for the prevention of GVHD. The degree of immunosuppressive activity required in the design of BMT conditioning regimens is dependent on the histocompatability of the bone marrow donor and patient, and the disease state being treated. Although autologous and syngeneic BMT may not require immunosuppressive activity, allogeneic BMT generally requires immunosuppressant activity. Increased immunosuppression may be necessary with increasing genetic disparity. Immunosuppression may be achieved with the use of radiation (TLI), cytotoxic agents (cyclophosphamide, procarbazine) or immunomodulators (cyclosporine, antilymphocyte/antithymocyte immunoglobulins, monoclonal antibodies). IX-A.

Total lympltoid irradiation (TLI)

Total lymphoid irradiation (TLI) employs the use of high-dose localized radiation of the lymphoid organs including the lymph nodes, thymus and spleen. Using this method of administration lymphocytes are progressively eliminated resulting in immunosuppression [ 1721. The effects of TLI on the immune system may include (1) direct lymphotoxic effects; (2) altered processing by immunocompetent cells within the lymphoid organs causing altered antigen processing; (3) the emergence of cells derived from non-T suppressor cells potentially capable of suppressing cell-mediated immune responses; and (4) an initiation of an immunotolerant state similar to the neonatal system [173, 1741. The resulting immune dysfunction provides a mechanism for immunosuppression of the host without the risk of sytemic side effects from high-dose irradiation. These effects have been useful for treatment of other disease states requiring immunosuppressive therapy including rheumatoid arthritis. Slavin et al. was one of the first to report the use of fractionated TLI as a immunosuppressive agent in conditioning regimens [l]. It is now commonly incorporated into BMT conditioning regimens for its immunosuppressive activity, especially in aplastic anemia. It can be administered as a single dose or fractionated in daily doses of 100-200 rads/day prior to BMT. Total lym-

phoid irradiation may also reduce the need for pharmacologic immunosuppression and may act synergistically with other immunosuppressive agents [172]. Ramsay et al. demonstrate that single fraction TLI is effective in the prevention of graft injection in aplastic anemia patients receiving conditioning with cyclophosphamide and TLI (7.5 Gy x I). Sustained engraftment was noted in 97% of patients with a history of multiple transfusions while patients receiving cyclophosphamide (200 mgkg) immunosuppression alone had only a 40% engraftment rate [ 1751. IX-B.

Cyclophosphamide

Cyclophosphamide is perhaps the most commonly used chemotherapeutic agent in BMT conditioning regimens. It is commonly employed in a variety of BMT conditioning regimens requiring immunosuppression and/or antineoplastic activity. Cyclophosphamide has been administered in combination with high-dose busulfan in Wiskott-Aldrich Syndrome, mucopolysaccharidosis, acute leukemia, acute myelofibrosis, Hodgkin’s disease, non-Hodgkin’s lymphoma and other refractory oncologic disorders [176-1781. It is used in combination with carmustine and etoposide in Hodgkin’s disease and in combination with TBI for leukemia, aplastic anemia and multiple myeloma [179]. It has also been used as a single agent in patients with severe aplastic anemia. Pharmacology and pharmacokinetics

Cyclophosphamide is an inactive prodrug which must be metabolized by the liver to its active species. The inactive parent drug is converted by hepatic microsomal enzymes to reactive intermediates (4-hydroxycyclophosphamide, phosphoramide mustard and nornitrogen mustard). The 4-hydroxycyclophosphamide metabolite plays a major role in the antineoplastic and immunosuppressive activities of cyclophosphamide. The 4-hydroxy metabolite is an active alkalating agent which inhibits rapidly dividing cells. The role of nornitrogen and phosphoramide mustard are less clear; however, they have been associated with the toxic effects of the drug [8]. Metabolism of cyclophosphamide to its active metabolites may be altered by agents which induce hepatic microsomal enzymes. Both cyclophosphamide and its metabolites appear to be distributed throughout the body, including the CSF. The pharmacokinetics of cyclophosphamide have been well described. Following administration of cyclophosphamide, only 10% of the parent compound is protein bound, while binding of its alkylating metabolites is on the order of 50%. Peak alkylating activity occurs in 2-3 hours after drug administration with a half-life

of 7.7 hours [180, 1811. Plasma half-life of the cyclophosphamide parent compound ranges between 4-7 hours. However, concentrations of the parent drug and its active metabolites can be detected in serum up to 72 hours after administration. Following repeat doses of cyclophosphamide the plasma half-life becomes progressively shorter, suggesting that cyclophosphamide may induce its own metabolism [9, 1821.Schuler et al. reported a reduction in cyclophosphamide half-life from 7.1 hours on day 1 to 4.3 hours on day 4 following a 4 day successive dosing schedule in patients receiving 50 mgkg x 4 prior to BMT. An accumulation of 4-hydroxycyclophosphamide and aldophosphamide with higher peak levels were also noted [ 1831.Whether these alterations are due to altered metabolism or saturation of cyclophosphamide protein binding sites remains unknown. Cyclophosphamide may accumulate with hepatic failure although dosing guidelines have not been set for the high doses required in BMT. Dosing guidelines in renal failure for standard dose therapy involves a 50% reduction in the dose and extension of the dosing interval from 12 to 18 hours with a GFR of ~10 mlimin. Immunosuppression

Cyclophosphamide is the major immunosuppressive agent used in bone marrow transplantation. It has been used as a conditioning agent to prevent acute graft rejection and as a post-transplant immunosuppressive agent. The immunosuppressive effects of cyclophosphamide are well described. Mullins et el. examined cyclophosphamide-induced immunosuppression in 12 patients receiving 120 mg/kg over a 2 day period. Immunosuppression as measured by antigen skin tests was reported in 50% of patients and lasted for a period of 2-4 weeks [184]. Cyclophosphamide selectively suppresses B lymphocyte function and lymphocyte functions which are mediated by T cells [185-1881. Cyclophosphamide is one of the most potent drugs used to suppress antibody response in mice [189]. Antihematopoietic activity

Cyclophosphamide is only a weak antihematopoietic agent. High- dose cyclophosphamide as a single agent has been used in doses up to 7 g/m2 (190 mg/kg) without BMT with hematologic recovery occurring within 3 weeks of cyclophosphamide administration [190, 1911. In vitro studies demonstrate that cyclophosphamide may inhibit committed stem cells and clonogeneic leukemia cells but is has limited effects on non-committed cells. Hagenbeek et al. compared the in vitro cytotoxity of the active 4-hydroxycyclophosphamide metabolite on normal hematopoietic stem and clonogenic leukemia

258

cells. Normal committed progenitor cells (CFU-C) were similar to clonogenic leukemia cells in their sensitivity to 4-hydroxycyclophosphamide (11 x 10m6M versus 9 x 10m6M), whereas non-committed CFU-S were less sensitive (55 x low6 M). A concentration of 50 x 1O-6M of 4-hydroxycyclophosphamide was found to eliminate all leukemic cells leaving behind a significant number of CFU-S to restore normal hematopoiesis [192]. Chang et al. report only a minor difference in sensitivity between normal and neoplastic cells. Exposure to 4-hydroxycyclophosphamide (29.2 &ml x 1 hour) resulted in elimination of 99.8% of HL-60 cells and 82.5% of CFU-GM [193].

atric patients and patients receiving continuous cyclophosphamide dosing. Hematuria may resolve spontaneously after discontinuation of therapy or may last for months. Agents such as MESNA which help to neutralize the active metabolite in urine have been used with some success. Non-reversible and sometimes fatal interstitial pulmonary fibrosis has been reported following high-dose therapy. An ‘SIADH-like’ syndrome may occur with high-dose cyclophosphamide therapy and may lead to progressive weight gain and hyponatremia. Other side effects include anorexia, nausea, vomiting, hepatic dysfunction, jaundice, dermatitis and hyperpigmentation of skin,

Antineoplastic activity Cyclophosphamide has activity in myeloproliferative and lymphoproliferate diseases, and has been used in non-Hodgkin’s lymphoma, sarcoma, lung, multiple myeloma, neuroblastoma and breast carcinoma. As a single agent used in patients with refractory solid tumors cyclophosphamide (120 mg/kg i.v. over 2 days) resulted in responses in 14/22 (11 PR, 3 CR) evaluable courses. In patients with lymphoid malignancies there was an overall response of 75% (58% PR, 17% CR). However, responses were of short duration (2-12 months) [ 1941. High-dose cyclophosphamide has also been examined as a single agent in BMT of small-cell lung cancer. Doses of 16&200 mg/kg prior to autologous BMT demonstrate an overall response rate of 81% (44% CR). Median survival time, however, was again short (69 weeks), following consolidation with irradiation (4000 rads). Santos et al. examined the antileukemic effect of high-dose cyclophosphamide (15&200 mg/kg) as a single agent. Although the treatment was well tolerated there was a 100% actuarial relapse rate of leukemia with no long term survivors [195].

IX-C. Cyclosporine A

Toxicity Fatal cases of diffuse hemorrhagic myocardial necrosis and acute myopericarditis have been reported in patients receiving > 180 mg/kg of cyclophosphamide, and in patients receiving cyclophosphamide in combination with carmustine, cytarabine and thioguanine [196198]. Cardiac damage may occur between 2 and 3 weeks after the start of therapy. Previous anthracycline therapy and concurrent chemo- or radiation therapy may predispose a patient to cyclophosphamide induced cardiac toxicities [197]. The exact dosing of cyclophosphamide may be very critical. Dosing of cyclophosphamide on a m2 basis rather than on a weight basis has reduced the incidence of cardiac toxicity. Severe hemorrhagic cystitis in 20% of patients receiving cyclophosphamide therapy has been reported with pedi-

Cyclosporine A is generally used as prophylaxis against GVHD; however, it has also been incorporated in BMT conditioning regimens for the prevention of acute graft rejection following BMT (particularly in severe aplastic anemia). Pharmacology and pharmacokinetics The pharmacology and pharmacokinetics of cyclosporine are complex. Cyclosporine has effects on many organ systems; however, its primary effect is on the immune system. Cyclosporine is metabolized in the liver and excreted in bile. At least 17 different metabolites have been identified [12, 1991. Following oral administration absorption is slow and incomplete. Approximately 37% of an oral dose is absorbed and peak blood concentrations occur in 24 hours. First pass metabolism of orally administered doses result in only 27% of a dose actually being available [12]. Following cyclosporine administration, cyclosporine distributes into blood and becomes bound to erythrocytes, leukocytes and lipoproteins. Less than 5% of cyclosporine remains free in plasma. Cyclosporine binds primarily to cholesterol containing lipoproteins. Cyclosporine concentrations are highest in the liver, endocrine glands, kidneys and adipose tissue. The distribution characteristics of cyclosporine are similar to those of the LDL receptor, It has been speculated that the LDL receptor may facilitate transport of cyclosporine across the cell membrane [200]. The volume of distribution of cyclosporine has been reported to be 1.3 liter/kg [12]. The pharmacokinetics of cyclosporine vary substantially between patients. The elimination half-life of cyclosporine has been reported to be in the range of 4-6 hours; however, altered hepatic and renal function significantly alter the pharmacokinetics of this agent. Kidney and liver function should be monitored closely, and the dose should be decreased in patients with reduced

259

renal or hepatic function [l 11. Therapeutic concentration ranges vary with the type of assay used, and therapeutic concentration ranges do not always correspond to response. Typically a therapeutic plasma concentration range of 5-200 r&ml as measured by radioimmunoassay, has been shown to produce satisfactory immunosuppression in most cases. However, some authors report that higher concentrations may be necessary. Bandini et al. report that concentrations of cyclosporine as measured by RIA should be >500 ng/ml in order to prevent acute GVHD [201]. However, concentrations of >500 @ml may also be associated with renal dysfunction. Immunosuppression

Cyclosporine selectively inhibits T-lymphocyte-dependent responses and can induce transplant tolerance in a variety of animal models [202-2041. Cyclosporine is known to inhibit the release of lymphokines that initiate cell-mediated immune response [205]. Marsh et al. examined the efficacy of cyclosporine in preventing graft rejection following allogeneic BMT in patients with severe aplastic anemia. Thirty-eight multiply transfused patients (2 10 units) received cyclophosphamide (50 mg/kg/day i.v. from day -5 to day -2) and cyclosporine starting on day - 1. Loading doses of 12.5 mg/kg BID; PO days -1 to +2, were followed by a maintenance dose of 6.25 mg/kg b.i.d.; days +3 onward. Cyclosporine was tapered over 6-12 months postBMT. Of 37 evaluable patients only 3/37 had initial graft rejection [206]. The incidence of graft rejection in multitransfused aplastic anemia patients with allogeneic BMT receiving cyclophosphamide alone is between 3060% [207]. The authors conclude that cyclosporine is effective in reducing acute graft rejection even after multiple transfusions. However, late graft rejection was still problematic and the authors suggest administration of full-dose cyclosporine for 9 months post-BMT with gradual reduction to discontinuation at 12 months [206]. Antihematopoietic and antineoplastic activity

Cyclosporine is specific for lymphocytes and does not appear to have significant antihematopoietic effects at the doses used in BMT. Cyclosporine has been noted to reverse multidrug resistance in a variety of cell lines in vitro, but it lacks significant antineoplastic properties as a single agent. Toxicity

Cyclosporine therapy has been associated with severe nephrotoxicity and extensive thrombosis of the glomerular arterioles and capillaries [208-2111. Nephrotoxicity has been associated with serum cyclosporine trough

concentrations of >500 @ml and concurrent use of aminoglycoside antibiotics [2 12, 2 131. Adequate hydration and mannitol diuresis may reduce the severity of nephrotoxicity. Therapeutic monitoring of cyclosporine plasma concentrations and dose adjusting may help to reduce the severity of nephotoxicity. How et al. used serum cyclosporine trough concentrations measured 12 hours after the dose to adjusted cyclosporine doses in aplastic anemia patients undergoing allogeneic BMT. Cyclosporine doses were initiated at (12.5 mg/kg b.i.d.; day -1 x 4 days, then 6.25 mg/kg b.i.d. x 6-8 months). Trough concentrations were maintained at 20&600 ngl ml and doses were reduced by 50% or discontinued if serum cretinine concentrations rose above 200 pmolil. Clinically severe nephrotoxicity was only noted in 3/21 patients who had received concurrent administration of aminoglycosides. Trough concentrations of cyclosporine were >lOOO ng/ml in all three. Other patients developed a mild, reversible elevation in plasma creatinine (100 to 205 pmolll [212, 21231. A variety of other side effects associated with cyclosporine have also been reported, including consumptive coagulopathy, hemolytic uremic syndrome, thrombocytopenia, and microangiopathic hemolytic anemia, hyperbilirubinemia, nausea, anorexia, tremor, hirsutism and pancreatic abnormalities [21 l-2141. Central nervous system toxicity has also been reported and may be associated with hypomagnesaemia [215]. Thompson et al. noted hypomagnesaemia (0.56 pmoli ml) in 7 patients with cyclosporine-induced grand ma1 seizures. Hypomagnesaemia is considered secondary to renal wasting of magnesium from cyclosporine induced renal tubular dysfunction. Seizures may be reversed by magnesium supplementation [2 161. IX-D. Procarbazine

Procarbazine is a methylhydrazine antineoplastic agent which has been used in conditioning regimens for aplastic anemia and congenital disorders. Procarbazine is primarily used as an immunosuppressive agent, and has been incorporated in BMT conditioning regimens to help prevent acute graft rejection. Pharmacology and pharmacokinetics

The antineoplastic activity of procarbazine is not fully understood but it appears to inhibit cells in S phase. Procarbazine distributes into most tissues including the liver, kidney, intestinal wall, CSF and skin. The parent drug undergoes hepatic metabolism to azo, azoxy and hydroxy derivatives. Agents which induce liver metabolizing enzymes, such as phenobarbital and

260

phenytoin, result in an increase in procarbazine clearance and decrease in peak azo concentrations [217]. Following oral administration procarbazine is almost completely absorbed with peak plasma procarbazine concentrations occurring in approximately 1 hour. Procarbazine has a short half-life (7 minutes) [218]. Approximately 2542% of a dose is excreted within 24 hours after administration [219]. Immunosuppressive activity Procarbazine causes aplasia of the lymphoid organs including the thymus [220]. Brent et al. report that procarbazine has only weak immunosuppressive activity as a single agent in mouse skin allograft studies; however, when combined with other immunosuppressive agents such as antilymphocyte serum (ALS) there is a profound synergistic action between agents [221]. The synergism between agents may be dependent on prior stimulation of the recipients immune system [222]. In patients with aplastic anemia who have been previously transfused, and are undergoing BMT with cyclophosphamide conditioning alone, there is a 30-60% graft rejection rate [207]. Conditioning regimens containing cyclophosphamide (50 mg/kg i.v.; days -5, -4, -3, and -2), ATS (0.2 ml/kg i.v. days -6, -4, -2) and procarbazine (12.5 mg/kg orally days -7, -5 and -3) have resulted in increased engraftment rates and graft rejection occurring in only 10% of patients and no incidence of late graft rejection [223]. Antihematopoietic activity Little information is available on the antihematopoietic effects of procarbazine at the doses used in BMT. Antineoplastic activity Procarbazine has significant activity in Hodgkin’s and non- Hodgkin’s lymphoma, glioblastoma and astrocytoma [224]. It has also shown some activity in myeloma, melanoma and oat-cell carcinoma of the lung. However, neoplastic activity is probably not significant at the doses used in conditioning regimens. Toxicity The dose-limiting side effects of procarbazine include mucositis, nausea, vomiting, central nervous sytem symptoms (somnolence, hallucinations, confusion, agitation, cerebellar ataxia) and peripheral neuropathy (paresthesia and myalgia). Other side effects have included photophobia, nystagmus, diarrhea, constipation, dermatitis, alopecia, pleural effusion and disulfiram-like reactions. Allergic reactions including skin rashes, pulmonary infiltrates, urticaria and eosinophilia are frequently reported and may be treated with corti-

costeroids [225]. Procarbazine is a weak MAO inhibitor and hypertensive reactions may occur with sympathomimetic agents, tricyclic antidepressants and foods high in tyramine. IX-E. Antithymocyte globulins

and

antilymphocyte

immuno-

Antithymocyte and antilymphocyte immunoglobulins (ATG and ALG) are derived from the serum of animals (rabbits, horses) hyperimmunized with human thymocytes/lymphocytes. The exact mechanism of immunosuppressive action is unclear. The cytotoxic effects are believed to be due to antibody/compliment induced lysis and possibly to opsonization with removal by the reticuloendothelial system. The specific cells lysed are the result of the particular immunization. In vitro studies suggest that ATG selectively depletes T-cells in peripheral blood, while ALG may act on a population of suppressor lymphocytes that inhibit normal stem cell growth and differentiation [22&228]. ATG binds to bone marrow cells including both erythroid and myeloid precursors and appears to be cytotoxic to committed macrophage progenitor cells, in the presence of complement. The pharmacokinetics of ATG and ALG have not been well characterized but are probably similar to the survival of the particular animal’s immunoglobulins. Following intravenous administration, ATG binds to its antigen and has a half-life between 1.5-12 days. ATG and ALG are used to decrease the incidence of acute immunological graft rejection and prevent the development of GVHD. These agents prevent graft rejection in solid organ transplants and are equally as effective as corticosteroids in the treatment of GVHD in HLA-identical sibling marrow grafts [4]. Immunoglobulins are associated with a variety of toxicities. Thrombocytopenia may be dose limiting and leukopenia is frequent [229]. Host immune reactions may result in hemolytic anemia, which may occur if anti-RBC antibodies are not absorbed. Fever and chills are seen in most patients and may be prevented with antihistamines. Serum sickness may be noted either during or after immunoglobulin therapy and may involve fever, malaise, arthralgia, nausea, vomiting, lymphadenopathy and cutaneous eruptions. Morbilliform rash may appear on the trunk, groin, axillae and extremities. Erythroderma and purpura have also been reported. Other side effects may include hypotension, azotemia, tachycardia, pulmonary edema, iliac vein obstruction, renal artery stenosis, diarrhea, stomatitis, hiccups, abdominal distention, dizziness, seizures, elevated liver function tests, hyperglycemia and paresthesias [227, 2291.

261

X. Monoclonal

antibodies (anti-LFA)

toxicity mg/m’

Monoclonal antibodies against the lymphocyte tional molecule LFA-1 (present on white blood

[57]. Combination

therapy

x 3) cyclophosphamide

with

BCNU

(120 mg/kg)

(150

and TBI

funccells)

(1000 rad) has resulted in acute liver failure [81]. Severe jaundice and liver failure has also been noted with VP-

have recently been identified and are currently being studied for their efficacy in preventing acute graft rejection in BMT conditioning regimens. Anti-LFA-1 has

16 (70 mg/kg) and TBI [ 131. The incidence of VOD from TBI may be increased with a high dose rate (1000 rad given at 23 rads/min) [233].

been shown to bind to LFA-1 and block immune activities in vitro and in vivo [230]. Fischer et al. evaluated the immunosuppressive activity of anti-HLFA-1 in

Nephrotoxicity. The addition of melphalan to combination conditioning regimens with cyclophosphamide, cis-

combination with busulfan and cyclophosphamide conditioning in patients with Wiskott-Aldrich syndrome,

which is not evident with the drugs given singly [234, 2351. The combination of high-dose melphalan and BCNU is not associated with increased renal toxicity,

Severe Combined Immunodeficiency and osteopetrosis. Following mismatched haploidentical BMT, all patients had rapid engraftment with leukocytes being of donor origin. Anti-HLFA-1

was well tolerated

with only tran-

sient fever (3840°C) after the first injection. The authors concluded that engraftment and hematologic recovery in patients receiving anti-LFA-1 was more rapid and complete than conditioning with busulfan, cyclophosphamide and ALG alone [231]. However, studies using this and other monoclonal antibodies as preparative agents remain to be fully evaluated. X-A.

Additive

toxicities

and drug interactions

Drug interactions and additive toxicities must be considered in the design of BMT conditioning regimens. Prior exposure to cytotoxic agents and the use of agents with overlapping toxicities may predispose patients to unnecessary toxicities. The selection of appropriate doses. scheduling of agents and monitoring of patients at risk for toxicities may help in reducing the potential for toxicity. Although little information exists regarding drug interactions at the very high doses used in BMT, a number of drug interactions and additive toxicities have been described. X-A. 1. Additive toxicities Pulmonary toxicity. Combination chemotherapy, particularly cyclophosphamide and methotrexate may increase the incidence of TBI-induced restrictive lung disease especially in the presence of infectious agents [232]. Patients receiving conditioning regimens with cyclophosphamide, VP-16 and BCNU have a significantly higher incidence of BCNU-induced interstitial pneumonitis, especially with high doses of BCNU (600 mg/m’)

WI. Hepatic toxicity. Hepatotoxic agents given prior to, or in combination with conditioning regimens may increase the risk of liver disease. Conditioning with AraC. BCNU or dimethylbusulfan in combination with other hepatotoxic agents may increase the risk of liver

platin and BCNU has resulted

although both and glomerular

in excessive renal toxicity

agents may have overlapping tubular toxicity when used in combination ther-

apy [86, 2361. In addition,

antibiotics

may increase

the

risk of nephrotoxicity to all other nephrotoxic agents, including cyclosporine. Morgenstern et al. report 13117 patients developing severe renal failure following conditioning with melphalan (140-250 g/m’) in combination with cyclosporin (12.5 mg/kg/day). Five of these patients required peritoneal dialysis. While both agents may cause renal impairment alone, the frequency and severity were much higher than expected from either agent [237]. Severe nephrotoxicity has also been associated with the concurrent administration of cyclosporine and aminoglycosides [2 I 21. Curdioto.ricity. Previous irradiation to the thorax or prior exposure to cardiotoxicity agents (anthracyclines) may increase the risk of cardiotoxicity by cyclophosphamide. Combination therapy with melphalan and cyclophosphamide have been noted to increase the incidence of cardiotoxicity. Electrocardiogram voltage abnormalities in 19123 patients and echocardiographic evidence of pericardial effusion was noted in 21% of patients receiving melphalan, cyclophosphamide and cisplatin. The high incidence of cardiac toxicity was assumed to be related to overlapping toxicity between melphalan and cyclophosphamide, since much higher doses of cyclophosphamide alone are necessary to produce cardiac toxicity [86]. Fatal, diffuse hemorrhagic myocardial necrosis and myopericarditis have been reported in patients receiving combination conditioning with BCNU, Ara-C and thioguanine [196- 1981. X-A.2. Drug inteructions Cyclosporine A (2 pug/ml) enhances the cytotoxic effects of VP-l 6 (3 pug/ml) in vitro and in vivo. Osieka et al. report an increase in the AUC of tritiated VP-16 by a factor of 1.5 in mouse leukemia and normal peripheral blood cells in the presence of cyclosporine. The antileukemic activity of VP-16 in Ll210 cells was also en-

262

activity at high concentrations. The cytotoxic effects for the two drug combination was higher than for either drug alone, especially at high concentrations [193]. In addition, since cyclophosphamide must undergo enzymatic activation by hepatic enzymes, drugs which stimulate or inhibit these enzymes may influence cyclophosphamide metabolism. The concurrent use of steroids may increase metabolism of cyclophosphamide necessitating an increase in cyclophosphamide dose. MTX may also inhibit the metabolism of cyclophosphamide to its active metabolites, potentially decreasing the activity of cyclophosphamide [242].

hanced by cyclosporine [238]. Since VP- 16 is highly protein bound, other agents which are highly protein bound may alter the unbound active fractions of VP-16. Evans et al. examined the effects of prior cytoxan exposure on radiation-induced damage to mouse bone marrow stem cells. The effects on survival of CFUs following cyclophosphamide (days 0 and 1) in combination with TBI on days 2, 3, 4, (6 fractions, twice daily, 0.08 Gy/min) was compared to cyclophosphamide and TBI alone (same dose). The combination treatment with cyclophosphamide and TBI resulted in an ‘overshoot’ of CFUs survival. Enhanced survival was noted between 11 and 23 days following initiation of treatment [239]. Other authors have reported a similar reduction in bone marrow lethality by TBI following cyclophosphamide drug treatment [240, 2141. While the mechanism of this interaction is not well understood it may be related to a drug-induced synchronization of stem cells into a phase of the cell cycle where they become relatively resistant to radiation [241]. The clinical significance of this interaction remains to be evaluated. Cyclophosphamide has also shown synergistic antitumor effects with other cytotoxic agents. Chang et al. examined the in vitro cytotoxic effects of 4-hydroxycyclophosphamide and VP- 16 on promyelocytic leukemia cells. The combination of 4-hydroxycyclophosphamide and VP-16 were reported to have synergistic cytotoxic

TABLE

XI. Discussion The design of BMT conditioning regimens is complicated by the multiple pharmacologic properties required to achieve successful engraftment and the potential toxicities associated with the cytotoxic agents used (Table 3). The primary consideration in designing an effective conditioning regimen must always include patient specific characteristics. These include disease state, type of transplantation, desired pharmacologic effects, potential toxicities, patient specific pharmacokinetics, and a pharmacokinetically based timing of transplantation. Successful engraftment following BMT is generally

4

The immunosuppressive,

antineoplastic,

and antihematopoietic

activities

of agents

used in BMT

Agent

Immunosuppression

Antihematopoietic

Antineoplastic

Ara-C

++

_

ATG Busulfan Carmustine

+++ _

_

Leukemia (esp. acute nonlymphoblastic _

+

+++ ++

Cyclophosphamide

+++

_

Cyclosporine Dimethylbusulfan Etoposide

+++ _

_

+

+++ ++

Melphalan

+

++

Procarbazine

++

_

Radiation Teniposide Thiotepa

++ ? +

+++ ++ +

Highly active (+++); moderate activity (++); minimal activity (+); not active (-). ALL, acute lymphocytic leukemia; ANLL, acute nonlymphocytic leukemia; CML,

Myeloid leukemias Multiple myeloma, gliomas, HodgkinWnon-Hodgkin’s lymphoma, mycosis fungoides Multiple myeloma, Hodgkin’s/ non-Hodgkin’s lymphoma, ALL ANLL. mycosis fungoides, solid tumors _ Ewing sarcoma, ALL, ANLL, CML Solid tumors, lymphoma, ANLL. ALL Solid tumors (breast, melanoma, sarcoma), multiple myeloma Hodgkin’s lymphoma, glioblastoma, astrocytoma Solid and hematologic Solid tumors (brain), lymphomas Solid tumors (breast, melanoma ovarian, lung, colon)

chronic

myelogenous

leukemia.

dependent on adequate antihematopoietic and immunosuppression activity. In addition, antineoplastic activity is often required. The degree of antihematopoietic, immunosuppressive and antineoplastic activity of some of the conditioning agents is summarized in Table 4. Combination therapy with cytotoxic agents and/or radiation are typically necessary to achieve adequate immunosuppression, antihematopoietic and antineoplastic activity. However, multidrug BMT conditioning regimens are often associated with drug interactions and overlapping toxicities between agents. Potential drug interactions which alter metabolism or elimination kinetics should be avoided or at least carefully considered. For example, patients who have received prior cisplatin therapy have reduced renal clearance of VP-l 6 and are prone to a higher incidence of VP-16 toxicity. The extent to which drug interactions effect the clinical efficacy of BMT regimens is often unclear and requires further investigation. In many cases overlapping toxicities between agents are predictable and can be avoided by chasing alternative agents or by closely monitoring susceptible patients. The use of specific agents to ablate unwanted side effects are being explored. Bianco and co-workers have studied

pentoxifylline, an agent capable of down-regulating tumor necrosis factor alpha which has been associated with the development of transplant-related complications. They observed in a small study a decrease in mucositis, hepatic venocclusive disease and renal failure [247]. These findings are being further studied including whether an increased relapse rate will occur. The pharmacokinetics of agents can also be monitored in order to avoid potential toxicities. Pharmacokinetic monitoring of high-dose regimens may assist in the identification of therapeutic concentrations of drugs and patients which may be at risk for lethal toxicities. Analysis of pharmakokinetics prior to BMT may also establish if newly transplanted cells may be exposed to residual cytotoxic concentrations of drug. For example, Holthuis et al. examined the serum pharmacokinetics of VP-16 after high-dose therapy and determined the terminal half-life is 8 hours with serum concentrations of up to 250 ng/ml being present 168 hours after infusion [14]. In vitro inhibitory concentrations of VP-16 are as low as 5 ,ug/ml when exposed for 1 hour. By back calculating, inhibitory concentrations of greater than 5 pug/ml are present 5 days after the end of a VP-l 6 infusion. Therefore, transplantation of new cells may have been

TABLE 5 Relationship between serum concentrations

and in vitro inhibitory activity

In vivo concentrations:

In vitro inhibitory concentrations:

Serum concentrations

Antihematopoietic

Drug

Dose*

C0nc.i time”

Ara-C [I, 125, 1271 Busulfan [5, 244, 2451 Carmustine [6, 7 I, 2461 4-Hydroxy cyclophosphamide [9, 1351 Etoposide [13,162] Melphalan 116, 901 Teniposide ~22, 231 Thiotepa [24, 1351

3 g/m’

1 pMi6 hrs

1 mgikg 800-1000 mg/m’ 5&60 mgikg

Inhib cont./ % inhibition

0.13 PM x 20 hrs/ >50% 0.2 PM/I 5 hrs 40.6 ,uM x 4 hrs 50% 56112 pM/30 min 93 PM x 1 hri >15% 4 pM/lO hrs lOOpMx1 hr/ 50%

30 mg/kg

3.3 pMl35 hrs

14@180 mg/m*

co.33 .uM/6 hrs

30&750 mg/m*

9.0 PM/IO hrs

45405 mg/m’

1.1-21.1 ,uM/8 hrs

activity

Antineoplastic activity

Cell line

Inhib cont./ % inhibition

CUF-GM

0.01 PM x 1 hr/ CFU-L 50% 400 PM x 2 hrsi Ll2lO >50% 9.0pM x 1 hr/50% Ll210

CFU-GM CFU-GM

Cell line

CFU-GM

33 PM x 1 hri >90%

MCF-7

17-34 FM x 1 hri 50% 100 PM x 8 hrs 50% -

CFU-S CFU-GM CFU-GM

EL-4

IO-25 PM x I hr/ 50%

CFU-GM

34 PM x 1 hri >90% 100 PM x 8 hrs/ >50% 0.02550.04 PM x 18 hrs/50% 140pM x 1 hri 90%

Neuroblastoma Osteosarcoma CEM MCF-7

Cell Lines: CFU-GM (colony forming units committed to granulocyte- macrophage differentiation); CFU-S (colony forming units-spleen); EL-4 (Murine T-cell leukemia-lymphoma); CEM (human lymphoblastic leukemia): MCF-7 (human breast cancer); CFU-L (leukemic colony forming units); Ll2lO (mouse lymphoblastic leukemia). *Doses administered; Ara-C: infusion over 3 hrs, twice daily x 6 days; Busulfan: oral every 6 hrs x 4 days; Carmustine: bolus over 10 min x l-2; 4-OH-cyclophosphamide: cyclophosphamide infusion over l-2 hrs/day x 24 days; Etoposide: infusion over 4 hrs x 1: Melphalan: bolus over 5 min x 1; Teniposide: continuous infusion over 72 hrs without BMT; Thiotepa: bolus over 1 min q day x 3 days. ‘* Serum concentrations measured at times following drug administration, concentrations reported are patient specific and are variable.

264

performed close to or during the time when inhibitory concentrations of VP-16 were present in plasma. Table 5 shows in vitro inhibitory concentrations for some of the agents used in BMT conditioning regimens. The pharmacokinetics of agents used in conditioning regimens are also dependent on patient specific characteristics. Patient variability in pharmacokinetics are due to a number of factors including altered absorption (of oral agents such as busulfan), altered distribution (obesity with BCNU), altered metabolism (microsomal enzyme induction with cyclophosphamide) and altered elimination of agents (as in age-related altered metabolic rates and decreased clearance). Pharmacokinetics may also be influenced by alteration of protein binding for highly bound drugs. The degree of protein binding for most agents has been determined; however, its influence on pharmacokinetics of agents remains to be determined in most cases. In general, for drugs which are highly protein bound, a reduction in protein concentration will result in an elevation of the ‘free’ unbound drug or fraction unbound. The fraction unbound represents the active fraction of a drug. Although it is tempting to suggest that patients with a low serum albumin resulting in an elevated fraction unbound should require a dose reduction, it is not that simplistic. Other variables including the type of protein the drug is bound to and the extent of metabolism of the free versus bound drug must also be considered. For instance, drugs with a high hepatic extraction ratio are cleared by the liver regardless of protein binding and liver metabolism is uneffected by the fraction of drug unbound. In this case an elevation in fraction unbound without altered metabolism may require a dose reduction. In contrast for drugs with a low hepatic extraction ratio, clearance is based on the unbound concentration. As the fraction unbound increases hepatic clearance increases and doses may need to be increased. Protein binding may also be influenced by other agents which compete for similar binding sites. Competition especially at the high doses used in BMT may result in elevated fraction unbound of one or both agents. Unfortunately, few studies have evaluated the free fraction of drugs used in conditioning regimens and how this correlates to albumin concentration. Further studies of the fraction unbound of high-dose single-agent therapy and multidrug regimens is necessary before dose adjustments can be recommended. Nevertheless, it is a reasonable hypothesis that significant improvement in the therapeutic ratio for high-dose multidrug BMT conditioning regimens could be obtained by careful patient-specific drug monitoring to assure that all individual patients receive the same drug dose and effect delivered to the target cells regardless of individual variance in metabolism, protein binding, and

kinetics of the drugs given. The technology for such monitoring has recently become available and could provide a means of testing this hypothesis. In summary, incomplete ablation of bone marrow due to inadequate or ineffective preparative regimens and regimen related toxicities are primary factors in BMT failures. An ideal preparative regimen must have complete ablative properties in addition to the creation of space for newly transplanted cells. Suppression of immune functions and immune mechanisms involved in graft rejection may also be required [243]. Antineoplastic activity may be necessary depending on the disease state. The design of a rational BMT conditioning regimen should also incorporate information gained from monitoring drug pharmakokinetics. Further evaluation of the pharmacokinetics of these regimens, especially in multidrug regimens, may lead to an improved understanding of the pharmacology of these agents. Since effective drug scheduling and dosing is based on all active species present following administration of agents, characterization of active metabolites is also essential. It is our belief, that with improved technological advances, and a better understanding of high-dose drug pharmacology, BMT conditioning regimens will eventually be tailored to the individual patient. Acknowledgements

Dr. Brian Smith is a Scholar of the Leukemia Society of America. We would like to thank Timothy B. Cadman for his help in preparing this manuscript.

Biographies V&vie Wiebe received her Pharm D from the University of California at San Francisco. She had a fellowship in the Section of Oncology at Yale University School of Medicine. She is currently an Assistant Professor of Medicine and Pharmacy at the University of Texas Health Science Center in San Antonio, Texas. Brian Smith received his AB degree from Princeton University and MD degree from Harvard Medical School. He is currently Associate Professor of Laboratory Medicine, Internal Medicine and Pediatrics at Yale University School of Medicine. He is the Associate Director of the Yale University Bone Marrow Transplantation Program. Michael DeGregorio also received his Pharm D degree from the University of California at San Francisco. At Yale University he was an Associate Professor in the Section of Medical Oncology. He is currently a Professor of Medicine at the University of Texas Health Science Center in San Antonio, Texas. Joel Rappeport received his AB degree from Yale University and his MD from Tuft University School of Medicine. Currently he is a Professor of Medicine and Pediatrics at Yale University School of Medicine and is Director of the Yale Bone Marrow Transplantation Program.

265

Reviewer This paper was reviewed by Massimo Martelli, M.D., Institute of Hematology, University of Perugia, Perugia, Italy.

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Conditioning regimens for allogeneic bone marrow transplantation.

Influence of nephrotoxic drugs on the late renal toxicity associated with bone marrow transplant conditioning regimens.

Selective decontamination in bone marrow transplant recipients.

Post-bone marrow transplant patient management.

Anaesthetic implications for bone marrow transplant recipients.

Successful bone marrow transplant for Fanconi's anaemia.

Reactivation of polyomavirus in bone marrow transplant recipients.

Pharmacokinetics of intravenous immunoglobulin (Gammagard) in bone marrow transplant patients.

Autologous bone marrow transplant in the treatment of acute leukaemia.

Normal cytogenetic values for bone marrow based on studies of bone marrow transplant donors.

Bone marrow transplant for a girl with bone marrow failure and cerebral palsy.

Catheter-related right-sided endocarditis in bone marrow transplant recipients.

Cytomegalovirus infection in the bone marrow transplant patient.

Chronic cyclosporine-associated nephrotoxicity in bone marrow transplant patients.

Multiorgan WU Polyomavirus Infection in Bone Marrow Transplant Recipient.

A marker chromosome in post-transplant bone marrow.

Defective neutrophil chemotaxis in bone marrow transplant patients.

Antisense agents in pharmacology.

Bone marrow transplantation in the busulfin-treated rat. III. Relationship between myelosuppression and immunosuppression for conditioning bone marrow recipients.

Long-term follow-up of bone marrow transplant patients.

New protective agents for bone marrow in cancer therapy.

Biomechanical comparison of the hand-based transplant used in bone-tissue-bone scapho-lunate ligament reconstruction.

MSC therapy attenuates obliterative bronchiolitis after murine bone marrow transplant.

Cyclophosphamide cardiotoxicity in bone marrow transplantation: a prospective evaluation of new dosing regimens.