Lack of Effect of Pentoxifylline Red Blood Cell Deformability Doyle

M. Cummings, and

PharmD, Mark

FCP,

J. Ellison,

FCCP, PharmD,

Samir

K. Ballas,

on MD,

FCP

The proposed mechanism of action for pentoxifylline’s beneficial effect in peripheral vascular disease is an improvement in red blood cell deformability. Likewise, single doses of pentoxifylline in healthy volunteers have been shown to improve whole blood filterability, which was suggested to occur as a result of augmented red blood cell deformability. To further assess this, the authors studied the effects of short-term pentoxifylline administration (400 mg three times daily for 7 days) on red blood cell deformability in ten healthy, methylxanthine-free, nonsmoking volunteers. Blood samples were obtained at baseline and after I week of therapy (steady-state). Samples were analyzed for red blood cell deformability by ektacytometry, which showed no significant change in deformability in any subject. Despite the improvement in whole blood filterability associated with both single-dose and short-term administration of pentoxifylline, the current study demonstrates no effect on red blood cell deformability after short-term administration in healthy volunteers.

p entoxifylline,

a methylxanthine derivative, is approved by the Food and Drug Administration for the management of intermittent claudication due to peripheral arterial disease. Its use produces modest improvement in walking distances relative to placebo in some patients.1-3 More recently, the drug has been used in a variety of other disorders, including acute renal failure, decubitus ulcer disease, cerebrovascular disease, and as an immunomodulator.47 The drug has been available in Europe since 1972, but the mechanism of action remains unclear. Because peripheral vascular disease has been shown to alter the flow properties of blood, it has been proposed that pentoxifylline may have beneficial effects that improve microcirculation. It has been suggested that pentoxifylline improves capillary blood flow by increasing red blood cell flexibility or deformability,

From the Department

of Family Medicine (Dr. Cummings), East Caroof Medicine, and the School of Pharmacy, University of North Carolina, Greenville, North Carolina; the Department of Medicine (Dr. Ballas), Cardeza Foundation for Hematologic Research, Jefferson Medical College, Philadelphia, Pennsylvania; and the Depart-

lina University

School

ment of Family Medicine (Dr. Ellison), East Carolina University School of Medicine and the School of Pharmacy, UNC, Greenville, North Carolina. Address for reprints: Doyle M. Cummings, PharmD, FCP, FCCP, East Carolina University School of Medicine, University of North Carolina, Greenville,

1050

5

NC 27858.

J COn Pharmacol

1992;32:1050-1053

therefore facilitating the movement of red blood cells through smaller capillaries. This theory is based on evidence that demonstrates enhanced filterability of red blood cells through a membrane of fixed pore size once the cells have been incubated with pentoxifylline.8’9 Improved whole blood filterability also has been demonstrated after single-dose administration of pentoxifylline both in healthy volunteers and in patients with peripheral arterial disease.10’11 This effect has been suggested to occur as a result of improved red blood cell deformability; however, there is little direct evidence to support this hypothesis. With respect to a mechanism of action, this proposed improvement in red blood cell deformability is suggested to occur as a result of inhibition of 3’,5’adenosine monophosphate diesterase, which in turn causes an increase in cyclic adenosine monophosphate in red blood cells. Again, there is little evidence to suggest that this mechanism occurs at drug concentrations that are achieved with usual therapeutic doses. As a result of these limited findings, the relationship between the drug’s hemorrheologic effects and patient outcome have been questioned.12 Moreover, our prior research demonstrates that red blood cells incubated with pentoxifylline or metabolite have no improvement in deformability as measured by ektacytometry, suggesting that some other mechanism may be operative.13 Because of the dem-

EFFECTS

onstrated improvement in whole after single doses of pentoxifylline teers, and the assertion that this proved red blood cell deformability, evaluate the effects of short-term pentoxifylline on red blood cell measured by ektacytometry.

OF

PENTOXIFYLLINE

blood filterability in healthy volunresults from imwe sought to administration of deformability as

METHODS Subjects

RBC

dynes/cm2 this study, cells was

(osmotic gradient ektacytometry). For the deformability index (DI) of red blood continuously recorded as the suspending medium’s osmolality was increased from 50 to 500 mmol/kg as previously described.15 The generated profile allows for determination of the maximum deformability index (Dlmax) or the DI at any desired osmolality (Figure 1). Baseline and treated samples were compared statistically using analysis of variance (ANOVA). RESULTS

Ten healthy volunteers, nine men and one woman, with a mean age of 30.4 years (±8.7 years) signed the informed consent statement approved by the University Institutional Review Board and completed the study. These subjects were not tobacco users, and agreed to avoid all methylxanthines, including caffeine, during the course of the study. Treatment All subjects received pentoxifylline 400 mg three times daily as a controlled-release tablet for 1 week (sufficient to achieve steady-state concentrations of both the parent compound and the metabolites). Compliance was monitored by pill counts; blood pressure, pulse, and any adverse reactions were monitored at the end of the 1-week study period. Samples

and

Analysis

At baseline and after 1 week of pentoxifylline administration, venous blood samples (6 mL) were collected in Vacutainer (Becton-Dickinson, Inc., Rutherford, NJ) tubes containing 1 mL anticoagulant acid citrate dextrose solution. Samples were mixed thoroughly after drawing and were stored under refrigerated conditions until analysis. Before analysis, the red blood cells were harvested, washed three times in isotonic buffer solution to remove the buffy coat, and then resuspended in calcium-free buffer solution to a uniform hematocrit. In a pilot study in this laboratory, three patients were studied after shortand long-term pentoxifylline administration, using both a whole-blood technique (without washing) and the current technique (with washing). The lack of difference between these two methods in the ektacytometric profiles suggested that washing had no impact on the assessment of drug effects. Prepared samples then were analyzed by ektacytometry for red blood cell deformability. The ektacytometer, a visco-diffractometer designed and previously described by Groner et al.,14 was used to measure whole-cell deformability in room air as a continuous function of the suspending medium’s Osmolality at a constantly applied shear stress of 170 i.r.a

ON

arn.

r.t’.,r.

a t,r.i,-t%

All ten subjects completed the study. Compliance with medication was greater than 90% of expected as assessed by pill counts. Normal red blood cells obtained from each subject at baseline produced a typical pattern of deformability (Figure 1). The DImax and the DI at 290 mmol (physiologic osmolality, DI2) were similar to those obtained previously in our laboratory from healthy volunteers and differed significantly from those obtained from patients with a variety of disorders that alter red blood cell deformability.13 After I week of treatment with pentoxifylline 1200 mg/day, there was no significant change in red blood cell deformability in any of the ten subjects (Table). Pentoxifylline was well tolerated in these subjects. One subject developed a headache, but it resolved despite continued therapy. There were no other adverse effects. DISCUSSION Vascular

disease

disorders

in the

___

remains United

Normal

one States

of the and

most

Europe.

common Although

RBC’a

I 300

200

Osmolality

Figure. Typical by ektacytometry.

pattern

of red blood

400

(MOSMOL/kg)

cell

deformability

as measured

CUMMINGS,

BALLAS,

TABLE Base lIne Subject

Number

Pentox ifylilne

DI,,,

DI

DI,,.

DI290

1

0.615

0.575

0.545

0.485

2 3

0.555 0.505 0.670 0.680 0.560 0.625 0.680 0.615 0.625 0.600

0.485 0.475 0.605 0.605 0.530 0.560 0.610 0.565 0.540 0.550

0.645 0.585 0.555 0.585 0.575 0.645 0.675 0.565 0.605 0.606

0.560 0.545 0.505 0.515 0.525 0.575 0.625 0.585 0.580 0.550

4

5 6 7 8 9 10 Mean

knowledge of the factors that affect microcirculatory flow is limited, it appears that the two most important factors are vessel radius and blood viscosity. Pentoxifylline has been shown to significantly reduce blood viscosity; however, the exact mechanism by which this occurs remains unclear. Early studies demonstrated that incubation of whole blood with pentoxifylline improved the filterability of blood through a filter of defined pore size (5-8 tm) similar to that of the capillary lumen.8’9 As noted above, these experiments have been replicated after singledose administration of pentoxifylline to both healthy volunteers and patients.10’11 This improved filterability has been assumed to occur as a result of increased red blood cell deformability. However, our previous research,13 as well as the current work, in which red blood cell deformability was evaluated by a standardized and reproducible methodology (i.e., ektacytometry), demonstrates no significant effect on red blood cell deformability in any patient. These findings suggest that one cannot assume that the improved erythrocyte filterability associated with pentoxifylline is the result of enhanced red blood cell deformability. As noted by both Marcel16 and Ely,5 a variety of factors affect red blood cell filterability and blood viscosity. Ektacytometry, as used in this study, has been used previously to evaluate the effects of various modalities on erythrocyte deformability.17’18 In particular, this technique is included in the review by Marcel16 as an important component of the evaluation of hemorrheologic agents. In previous studies of the drug monensin, ektacytometry was used to characterize the effects of the drug on red blood cell deformability.18 This technique has also been used to evaluate potential drugs for sickle cell disease19 and may provide more information into the skeletal protein interactions that affect deformability. 1052

5

.i ClIn Pharmacol

1992;32:1050-1053

AND

ELLISON

From the results of our earlier work in vitro,’3 we could not rule out the possibility that improved red blood cell deformability would occur after pentoxifylline treatment in volunteers. Further, because only the parent compound and metabolite 1 were available for study in vitro, we could not rule out the possibility that one of the other metabolites of pentoxifylline formed after administration in humans would result in improved red blood cell deformability. This study addresses both issues and demonstrates a consistency between our prior work in vitro and the current work with healthy volunteers. Because this study was only 1 week in duration and involved only healthy volunteers, however, we cannot rule out the possibility that shortor long-term administration of pentoxifylline in patients with peripheral vascular disease may improve red blood cell deformability. In the current study, volunteers received pentoxifylline for I week to ensure that serum concentrations of both the parent compound and all endogenous metabolites had reached steady state. Despite the presence of all metabolites in physiologic concentrations, no red blood cell deformability was observed. This suggests that, just as with the hydroxyhexyl metabolite 1, other metabolites also would not have demonstrated activity in vitro.13 These findings cast doubt on the assumption that improved whole blood filterability occurs as a result of improvement in red blood cell deformability. Further, they support the hypothesis that the therapeutic effects of pentoxifylline are not the result of red blood cell deformability, but are related to some other mechanism. This topic is the subject of investigation by a number of researchers. Earlier studies focused on the red blood cell, but more recent attention has shifted to the effects of the drug on leukocyte and immune function. Recent research suggests that microvascular flow may be influenced by a complex interaction of white blood cells and platelets with red blood cells and the endothelium.’#{176} In this regard, pentoxifylline has been shown to affect the filterability of white blood cells as well as the aggregation of platelets. The drug also has been shown to diminish margination of white blood cells, and its use in septic shock and acute respiratory distress syndromes is being considered.21-23 The mechanism responsible for the effects in leukocytes is presently being worked out2426; however, discrepancy between work in vitro and in vivo has hampered progress.27 Another important body of research demonstrates that pentoxifylline can inhibit the action of interleukin-I and tumor necrosis factor7 and may be beneficial as an immunomodulator. Finally, recent evidence demonstrates that pentoxifylline does not produce blood lipid changes and that increased

EFFECTS

OF

PENTOXIFYLLINE

filterability cannot be attributed to a reduced tent of membrane cholesterol or to an increased portion of polyunsaturated fatty acids in the brane.28

conpromem-

In contrast to the improvement in red blood cell filterability associated with pentoxifylline both in vitro and after single doses in normal volunteers and patients, the current study demonstrates that pentoxifylline, administered in usual doses for 1 week to healthy volunteers, has no significant effect on red blood cell deformability as measured by ektacytometry. Future research should attempt to define the mechanism or mechanisms of drug action by studying the factors that regulate microvascular flow.

sel

Pharmaceutical

Company

for

Ms. Mary Dent for editoand the Hoechst-Rous-

supplies

11. Ambrus Stadler

claudication:

BY, Reich efficacy

Multicenter,

controlled,

lite

on

try.

Angiology

red

blood

14.

in

trial

arterial

disease

the treatment placebo-controlled

Clin

Chem

tiers of Pharmacology: of Limb Ischemia. phia, 1983;3:125-140. Chasis

JA.

Mohandas

N:

Two

Clark

MR,

sodium

19.

RM,

Johnson

20.

by

Feo

CJ,

Schmid-Schonbein

teraction

CL:

6.

Hsu

CY,

and

its

Norris

in

1W,

acute

cebo-controlled 7.

Sullivan

on

the

skin.

Dermatol

Hogan

EL,

Bladin

nonhemorrhagic

Carper

P. Dinsdale

stroke:

trial.

HT,

Stroke

Novick

WJ,

CL:

8. Ehrly

of pentoxifylline

effect

9. Sowemimo-Coker filterability of normal cultured endothelial

red cells.

Grigoleit HG, Porsche effect of pentoxifylline subjects after administration 1976:1:241-247.

HEMATOLOGIC

Medical

SO, Turner

10.

AGENTS

Inhibition

of

necrosis facInfect Immun

on the flow properties

Science

P: The

1975;

effect

of pentoxifylline

blood cells and their Eur J Clin Pharmacol E, Stefanovich on red blood of Trental

of

3:465. adhesiveness

on to

1985; 29:55-59.

V. Jacobi C, Lahham A: cell flexibility in healthy 400. Pharmatherapeutica

M, Dobo

and

Usami

cells

Invest

I: Evaluation

scan

are indeJ Cell Biol

of sickle

1982;

of covalent

ektacytometry.

Blood

5, Skalak R, Chien S: The inin capillary and postcapil-

Effects

of pentoxifylline

on endo-

protein accumulation in Mandell C, Novick

dog,

Function.

New

York:

and neutroW (eds.): Pen-

Haber

and

Flora,

190-198.

Josaki

K, Contrino

Mandell

J, Kristie

ARDS,

J, Krause

of human neutrophils.

P. Kreutzer leukocyte

and

DL: Pentoxi-

function

in vitro.

pentoxifylline.

Am

Rev

Dis 1988;138:1103-1105.

24. Rao KMK, Crawford J, Currie ization and inhibition of capping man 582.

SB: Hydration J Clin

P02

D, WeiIJV:

Leukocyte

lymphocytes

and

neutrophils.

MS. Cohen HJ: Actin depolymerinduced by pentoxifylline in huJ Cell Physiol 1988;137:577-

25. Needham D, Armstrong M, Hatchell DL, Nunn RS: Rapid formation of “passive” polymorphonuclear leukocytes: The fects of pentoxifylline. I Cell Physiol 1989;140:549-557. 26. Cohen

ICRS

The

pla-

1988; 19:716-722. Mandell

of interleukin-i and tumor function by pentoxifylline.

blood.

Yatsu

a randomized,

the inflammatory action tor (alpha) on neutrophil 1988; 56:1722-1729. AM: The

HB,

P. Caplan LR. Karp HR. Swanson PD, Mayman Cl, Cobert B, Savitsky JP: Pen-

double-blind GW,

hyperosmolar

Gun

Microvasc CH, Lien

toxifylline

Ilespir

FM, Earnest MP, Scheinberg Faldman RG, Cohen MM, toxifylline

effects

associations.

erythrocytes lIes 1980;19:45-70.

23.

ized hyperviscosity 1988; 6: 585-608.

Nossal

and

extravascular in the

22.

deformabil-

that

monensin.

GW,

of leukocytes

toxin-induced phil sequestration 1988;

of local-

for

ektacytometer.

66:432-438.

modulation 1990;136:623-630.

A review

technique

the

properties

protein

by

fylline-induced Am J Pathol

in dermatology:

ektacytome-

membrane

membrane

compounds

Pharm

therapy

JM,

Dose-re-

Approach to the Treatment of Physicians of Philadel-

N, Shohet

vessels.

5(10)(Suppl):S-150.

by

optical

with

Erythrocyte

ionophore

Welsch

H: Pentoxifyllmne

Anain JP:

filterability: CliniTher 1990; 48:50-

measured

M: New

skeletal

Mohandas

using the 70:1074-1080.

anti-sickling

of interad-

as

distinct

21.

Ely

PM,

Savitsky

on erythrocyte Clin Pharmacol

Pharmacologic Philadelphia: College

lary

5.

BL,

15. Clark MR. Mohandas N, Sholet SB: Osmotic gradient ektacytometry: Comprehensive characteristics of red blood volume and surface maintenance. Blood 1983; 61:899-910. 16. Marcel GA: Hemorrheologial Agents, in Symposia on Fron-

1984; 35:396-406.

1988;

Anain

Cobert

cell deformability 1990;41:118-123.

3. Strano A, Davi C, Avellone C, Novo S. Pinto A: Double-blind, crossover study of the clinical efficacy and the hemorrheological effects of pentoxifylline in patients with occlusive arterial disease of the lower limbs. Angiology 1984; 35:459-466. 4. Vadiei K, Iyer LV, Brunner U. Luke DR: Study of pentoxifylline in experimental acute renal failure (ARF) (Abstract PP1208). lIes

SM,

JA,

W, Mohandas N, Besis erythrocyte deformability 1980; 26:1435-1442.

Croner measuring

1985;

2. Roekaertis F. Deleers L: Trental 400 mittent claudication: Results of long-term,

Angiology

double-blind

occlusive

Anain

12. Johnson WC, Sentissi JM, Baldwin D, Hamilton J, Dion J: Treatment of claudication with pentoxifylline: Are benefits related to improvement in viscosity? I Vasc Surg 1987:6:211-216. 13. Cummings DM, Ballas S: Effects of pentoxifylline and metabo-

ity and stability:

T, Reichle FA, Scogin JT, in the treatment of inter-

assessment of chronic Heart! 1982;104:66-72.

ministration.

JM,

P. Brobst

sponse effects of pentoxifylline cal and animal model studies.

18.

with objective patients. Am

Anain

pendently regulated 1986; 103:343-350.

JM. Cutler BS, Lee DE: Pentoxifyllmne

mittent

JL,

5, Mitchell

17.

of pentoxifylline.

REFERENCES 1. Porter Strandness

RBC

56.

CONCLUSION

The authors thank Ms. Jerri Harris and rial and statistical assistance, respectively,

ON

Currie HJ:

MS. Rao

KMK.

Stimulus-specific

Padmanabhan effects

phil CR3 expression, degranulation Leukoc Biol 1990;147:244-250. 27. Rao KMK, Simel DL, Cohen of pentoxifylline

administration

cytoskeletal function in patients Lab Clin Med 1990:115:738-744. 28. Clemens membrane 624.

MR. Ruess and plasma

J, Jones

with

blood

on

superaxide

HJ, Crawford on

A, Crawford

of pentoxifyllmne

and

intermittent

M: Effect of pentoxifylline lipids. Eur J Clin Pharmacoi

J,

neutro-

production.

J, Currie viscosity

deef-

I

MS: Effects

and

leukocyte

claudication. on

erythrocyte 1991:41:623-

1053