Rheumatology International

https://doi.org/10.1007/s00296-018-3995-3
Rheumatology
INTERNATIONAL

CLINICAL TRIALS

Comparing the efficacy of low-dose vs high-dose cyclophosphamide

regimen as induction therapy in the treatment of proliferative lupus
nephritis: a single center study
Sonal Mehra1 · Jignesh B. Usdadiya1 · Vikramraj K. Jain1 · Durga Prasanna Misra1,2 · Vir Singh Negi1

Received: 30 December 2017 / Accepted: 7 February 2018

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Cyclophosphamide (CYC) has been the backbone immunosuppressive drug to achieve sustained remission in lupus nephritis
(LN). The aim was to evaluate the efficacy and compare adverse effects of low and high dose intravenous CYC therapy in
Indian patients with proliferative lupus nephritis. An open-label, parallel group, randomized controlled trial involving 75
patients with class III/IV LN was conducted after obtaining informed consent. The low dose group (n = 38) received 6 ×
500 mg CYC fortnightly and high dose group (n = 37) received 6 × 750 mg/m2 CYC four-weekly followed by azathioprine.
The primary outcome was complete/partial/no response at 52 weeks. The secondary outcomes were renal and non-renal
flares and adverse events. Intention-to-treat analyses were performed. At 52 weeks, 27 (73%) in high dose group achieved
complete/partial response (CR/PR) vs 19 (50%) in low dose (p = 0.04). CR was higher in the high dose vs low dose [24 (65%)
vs 17 (44%)], although not statistically significant. Non-responders (NR) in the high dose group were also significantly lower
10 (27%) vs low dose 19 (50%) (p = 0.04). The change in the SLEDAI (Median, IQR) was also higher in the high dose 16
(7–20) in contrast to the low dose 10 (5.5–14) (p = 0.04). There was significant alopecia and CYC-induced leucopenia in
high dose group. Renal relapses were significantly higher in the low dose group vs high dose [9 (24%) vs 1(3%), (p = 0.01)].
At 52 weeks, high dose CYC was more effective in inducing remission with decreased renal relapses in our population.
Trial Registration: The study was registered at http://www.clint​rials​.gov. NCT02645565.

Keywords Systemic lupus erythematosus · Lupus nephritis · Cyclophosphamide · Adverse effect · Efficacy

Abbreviations
ACR​ American College of Rheumatology
Electronic supplementary material The online version of this ALMS The Aspreva Lupus Management Study
article (https​://doi.org/10.1007/s0029​6-018-3995-3) contains
CBC Complete blood count
supplementary material, which is available to authorized users.
CYC​ Cyclophosphamide
* Vir Singh Negi CR Complete response
vsnegi22@yahoo.co.in C3, C4 Complement component 3 and 4
Sonal Mehra dsDNA Double-stranded deoxyribonucleic acid
drsonalmehra@gmail.com eGFR Estimated glomerular filtration rate
Jignesh B. Usdadiya ELISA Enzyme-linked immunosorbent assay
jigneshusdadiya@gmail.com ELNT Euro Lupus Nephritis Trial
Vikramraj K. Jain HPF High power field
vikramraj.jain@gmail.com IV Intravenous
Durga Prasanna Misra ISN/RPS International Society of Nephrology/Renal
durgapmisra@gmail.com Pathology Society
1 ITT Intention to treat
Department of Clinical Immunology, Jawaharlal Institute
of Postgraduate Medical Education and Research (JIPMER), IQR Interquartile range
Puducherry 605006, India LN Lupus nephritis
2
Sanjay Gandhi Postgraduate Institute of Medical Sciences, LDL-C Low-density lipoprotein cholesterol
Lucknow 226014, India MDRD Modification of Diet in Renal Disease

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Vol.:(0123456789)

MMF Mycophenolate mofetil
NR No response
NIH National Institutes of Health
PR Partial response
RBC Red Blood cell
SD Standard deviation
SLEDAI Systemic Lupus Erythematosus Disease Activ-
ity Index
WBC White blood cell
Introduction
Lupus nephritis (LN) develops in 50–60% of patients during
the first 10 years of onset of systemic lupus erythematosus
(SLE), and it increases the risk of renal failure, cardiovascu-
lar disease, and death [1]. The target of immunosuppressive
therapy in LN is to produce sustained renal remission with-
out increasing drug toxicity. Recent meta-analyses suggest
that mycophenolate mofetil (MMF), calcineurin inhibitors,
or their combination were most effective for inducing remis-
sion compared to intravenous cyclophosphamide (CYC),
while conferring similar or lower adverse event profile [2,
3]. However, in a lower–middle income country such as
India, due to limited resources, cyclophosphamide is a more
cost-effective option than MMF or calcineurin inhibitors.
Although MMF is an equally effective treatment in young
females as the preservation of fertility is not an issue as is
with cyclophosphamide, however, in our experience, in a
resource-poor country such as India, very few patients are
able to afford the cost of MMF therapy for a period of 6
months, therefore, resulting in lesser compliance. Long-term
follow-up studies have shown that intermittent pulse intra-
venous CYC therapy in combination with glucocorticoids is
effective for moderate to severe proliferative LN [4–6]. The
initial regimen advocated for the treatment for proliferative
LN by the National Institute of Health (NIH) [7] and further,
the modified NIH protocol that comprised of six monthly
pulses of 0.5–1gm/m2 of IV CYC for remission induction
followed by azathioprine as maintenance therapy. Although
a longer course of CYC was more effective in preventing
disease flares, it was associated with significant toxicity [7].
The low-dose Euro LN Trial (ELNT) and its 10-year follow-
up data demonstrated a comparable efficacy and safety pro-
file of low-dose and high-dose CYC [5, 8] in Caucasians. It
has been observed that more severe forms of LN are more
frequent in African-Americans, Asians and Hispanics from
Mexican ancestry compared to Caucasians [9–11]. Previous
studies have noted that, despite the use of CYC, 18–57%
of patients may fail to achieve remission [12]. The present
study aimed at identifying the optimal dose, frequency, dura-
tion and safety of induction therapy of two different CYC
options in subjects with proliferative Class III/IV LN. We
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Rheumatology International
hypothesized that low-dose CYC and high-dose CYC had
similar efficacy as induction therapy in the treatment of pro-
liferative LN. Our study is the first study on our population
that scrutinizes the efficacy and risks of both these treat-
ments protocols in our environmental and genetic makeup.
Materials and methods
Trial design
This study was an investigator-initiated, open label, parallel
group randomized controlled trial at the Jawaharlal Institute
of Postgraduate Medical Education and Research (JIPMER),
Puducherry, a major centrally-funded tertiary-care referral
center in South India, from December 2015 to December
2016. All LN patients who met inclusion/exclusion crite-
ria were enrolled in the study after taking written informed
consent.
Inclusion criteria
1. Diagnosis of SLE according to the American College of
Rheumatology criteria [13]
2. Age > 16 years
3. Proteinuria ≥ 500 mg in 24 h and/or urine routine
microscopy showing active cellular casts/sediments [> 5
RBC/high power field (HPF) and > 5 WBC/HPF and
cellular casts]
4. Biopsy-proven proliferative lupus glomerulonephritis of
class III, IV according to the International Society of
Nephrology/Renal Pathology Society (ISN/RPS) criteria
[14]
Exclusion criteria
1. Patients ever treated previously with intravenous or
oral cyclophosphamide, mycophenolate mofetil, cyclo-
sporine or steroids > 15 mg/day in the last 3 months.
2. Patients with renal thrombotic microangiopathy, pre-
existing chronic renal failure, pregnancy, previous
malignancy (except skin and cervical intraepithelial
neoplasia), diabetes mellitus or coronary heart disease.
3. Patients with previously documented severe toxicity to
immunosuppressive drugs.
4. Patients with active acute or chronic infections
5. Pregnancy
Baseline evaluation
Demographic details including age, sex and disease dura-
tion; history of renal disease and treatment with other immu-
nosuppressive drugs, systemic feature, comorbidities, side
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effects of therapy at each visit and disease activity—SLE
disease activity index (SELENA-SLEDAI) and renal SLE-
DAI. The laboratory evaluation included complete blood
count (CBC), renal function tests (RFT)/estimated glomer-
ular filtration rate (eGFR) by the Modification of Diet in
Renal Disease (MDRD) equation, liver function tests (LFT),
urine routine and microscopy, urine protein creatinine ratio
(U-PCR), complement component 3 (C3) and 4 (C4) by
nephelometry, anti-dsDNA (double-stranded DNA) antibody
by enzyme-linked immunosorbent assay (ELISA). Patho-
logical assessment of renal biopsy was performed using the
International Society of Nephrology/ Renal Pathology Soci-
ety 2003 classification system with an assessment of active
and chronic glomerular and tubulointerstitial changes with
activity and chronicity scores [15].
Interventions
All patients received three daily pulses of 1 g intravenous
methylprednisolone followed by 1 mg/kg/day of predniso-
lone for 4 weeks tapered by 5 mg every 2 weeks to reach a
dose of 5–7.5 mg/day until completion of 52 weeks. The
maximum dose of prednisolone was 60 mg/day. In the low-
dose CYC regimen, patients received six fortnightly IV CYC
cycles at a fixed dose of 500 mg, while the high-dose CYC
regimen comprised of four weekly six cycles of 750 mg/
m2 with a maximum of 1.5 g per pulse. After completion
of induction, oral azathioprine 2 mg/kg was started two
weeks after the last CYC dose. For patients with azathio-
prine-related toxicity, the dosage was reduced to 1 mg/kg/
day. In the case of use of rescue therapy (MMF/rituximab/
intravenous immunoglobulin and/or high-dose corticoster-
oids administered for > 2 weeks), patients were discontinued
from the study and classified as a non-responder.
Concomitant treatment
All patients received hydroxychloroquine during the study
(5–6 mg/kg, 400 mg/day maximum) after normal baseline
fundus evaluation. Hypertension (diastolic blood pressure
> 90 mm Hg) was treated with angiotensin-converting
enzyme inhibitors (unless contraindicated) and other appro-
priate drugs. Atorvastatin was started for patients with low-
density lipoprotein cholesterol (LDL-C) > 100 mg/dl. Cot-
rimoxazole prophylaxis was given for Pneumocystis jiroveci
pneumonia. Use of non-steroidal anti-inflammatory drugs
was prohibited due to their potential for nephrotoxicity.
Primary outcome measures
Renal response as defined by the European League against
Rheumatism (EULAR) guidelines [16] was evaluated at
52 weeks. Inactive urinary sediments were defined by ≤ 5
red blood cells (RBC)/ HPF, ≤ 5 white blood cells (WBC)/
HPF and no cellular casts as per the American College of
Rheumatology (ACR) definition [17]. We also compared
renal responses at 12 and 24 weeks.
Complete response (CR): defined as urine protein cre-
atinine ratio (UPCR) < 0.5 g and normal (GFR > 90 ml/
min) or stable (< 10% deterioration from baseline if GFR
was previously abnormal) renal function and inactive uri-
nary sediments.
Partial response (PR): defined as ≥ 50% reduction in
proteinuria to sub-nephrotic levels, normal (GFR > 90 ml/
min) or stable (< 10% deterioration from baseline if GFR
was previously abnormal) renal function and inactive uri-
nary sediments.
No response (NR): Patients were classified as non-
responders if criteria for CR or PR were not met and or if
they experienced severe flare.
Secondary outcome measures
Proportion of patients with renal and non-renal disease
flares
Classification and treatment of flares
• Benign non-renal flare (i.e. those not meeting the defini-
tion of severe flares) was treated with low-dose predni-
solone (15 mg of prednisolone /day) for 2 weeks period.
• A severe flare was defined as below-
• Severe systemic disease was defined as any of the fol-
lowing events: central nervous system involvement due
to lupus, fall in platelet count (< 100,000 platelets/mm3),
hemolytic anemia, lupus pneumonitis/myocarditis, exten-
sive vasculitis of the skin or serositis not improving with
low-dose corticosteroid therapy.
• Nephritic flares were defined as reproducible elevation
of serum creatinine by 30% or more (or a reduction in
GFR at least 10%) along with active urine sediments with
an increase in glomerular hematuria by 10 or more red
blood cells per high power field, irrespective of whether
changes occurred or not in urine protein to creatinine
ratio (U-PCR). Proteinuric flares were defined as repro-
ducible doubling of U-PCR > 1.0 after having attained
complete renal response or > 2.0 after previously attain-
ing partial response.
• A severe flare was treated by rescue therapy, and such a
patient was considered to be a non-responder.
Assessment of adverse events: patients were monitored
for adverse events and infections every two weeks for the
low-dose group and every four weeks for the high-dose
group.
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Sample size
The sample size was determined using the sample size
calculator version 2 software (nMaster 2.0) to determine
the sample size for a two arm, randomized, parallel group
trial with the outcome variable being binary and the objec-
tive of showing non-inferiority. Assuming a response rate
of 80% with high-dose regimen [18] and 60% with low-
dose CYC [8] and the non-inferiority criteria of 20%, it
was estimated that to achieve 80% power with one-sided
confidence interval of 95%, accounting for a withdrawal
rate of 10%, 75 subjects needed to be recruited.
Randomization
Patients were randomized, using block randomization,
eight blocks of 10 patients each with 1:1 random alloca-
tion was performed in 75 LN patients to low-dose nephritis
protocol of six fortnightly pulses at a fixed dose of 500 mg
for 12 weeks or a high-dose CYC regimen comprising of
six doses four weekly of 750 mg/m2 using a computer-
generated random number table. A fellow researcher had
given random block and number to patients sequentially,
who was unaware of treatment allocation and had no other
role in the study. The trial was open-label and unblinded
to reflect a real-life clinical practice scenario. The insti-
tutional ethics committee (human studies) at JIPMER
approved the study.
Statistical methods
Continuous data were presented as mean ± standard devia-
tion (SD) or median with interquartile range (IQR), as
appropriate. Qualitative or categorical variables were
described as frequencies and proportions. The Kolmogo-
rov–Smirnov test checked the normality of the quantita-
tive data. For normally distributed data, means for the two
groups were compared using Student’s t-test. Proportions
were compared using Chi-square (χ2) or Fisher’s exact test
as applicable, and medians were compared using a non-
parametric test. For patients who were lost to follow-up
(including mortality/adverse events /treatment default), the
last observations were taken as their outcome for intention
to treat (ITT) analysis. All calculations were performed
using SPSS version 21. The analysis was based on the
intention-to-treat principle, regardless of treatment adher-
ence. p < 0.05 was set as threshold for significance.
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Results
A total of 101 subjects were screened (depicted in Fig. 1).
Baseline characteristics of the study subjects
and renal parameters
The baseline characteristics of the study population and real
prarameters are described in Tables 1 and 2 respectively.
There were 38 study subjects in the low-dose and 37 in
the high-dose group. The mean age of onset of nephritis
(mean ± SD) in the low dose vs high dose (30.71 ± 10.04 vs
27.24 ± 10.60 years) and disease duration in months (median
IQR), 18 (4–36) vs 12 (3.5–24), were similar. The median
cumulative dose of CYC in grams in the low-dose group
was 3 (3–3) grams and the high-dose group was 6 (5.4–6.3)
grams (p < 0.0001). All the baseline clinical features and
organ involvement present concurrently at the time of lupus
nephritis in the low-dose and high-dose groups were simi-
lar, except leucopenia, which was significantly higher in the
low-dose [14 (34%)] compared to the high-dose group [5
(14%), p = 0.02].
Primary response outcomes
At the completion of 52 weeks, the patients achieving com-
plete/partial response (CR/PR) in the high-dose compared
to the low-dose group were significantly higher (p = 0.04),
as depicted in Tables 3, 4. The renal responses were also
assessed at 12, 24 weeks as depicted in supplementary
Fig. 1. We observed that at the completion of 12 weeks,
the patients achieving complete response (CR) in the low
dose compared to the high dose were significantly higher
18 (47%) vs. 9 (24%) (p = 0.03) and complete or partial
renal response were 23 (60%) in the low vs 11 (30%) in the
high dose (p = 0.01). However, at 24 weeks, the primary
responses and renal SLEDAI were similar. We also analyzed
their autoantibody profile, which was similar in both the
groups.
Kinetics of response to therapy
The kinetic of response to therapy with respect to urine
PCR, serum albumin, Renal SLEDAI, complements,
anti-dsDNA and SLEDAI are depicted in supplemen-
tary Figs. 2–8 (Assessment of Secondary outcomes). The
secondary outcome measures included adverse events
and disease flares as summarized in Table 5. The total
numbers of deaths were equal in both the treatment
arms, being 2(5%) in the low dose vs 2(5%) in the high-
dose group. In the low-dose group, one subject expired
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Fig. 1  Disposition of the study

participants. CYC​cyclophos-
phamide, ITT intention to treat,
LN Lupus nephritis, MMF
mycophenolate mofetil

following gram-negative sepsis and while the second
case succumbed to H1N1 swine flu with acute respira-
tory distress syndrome. In the high-dose CYC arm, both
the study subjects expired secondary to severe leucopenia
(one developed pneumonia and gram-negative sepsis; the
other developed abdominal wall cellulitis and multi-organ
failure). In the low-dose group, one developed gastroin-
testinal vasculitis during induction therapy that required
rescue therapy with high-dose CYC and one developed
thrombotic thrombocytopenic purpura in the high-dose
group during maintenance. Renal relapses were signifi-
cantly higher in the low dose being 9(24%) vs high dose
1(3%) (p = 0.01). The median time to relapse in weeks was
similar in both groups during the follow-up period [median
with IQR 48(41–50) weeks in the low-dose CYC group vs
46 (40–52) weeks in the high-dose CYC group, p > 0.99].
Discussion
The results of the trial indicate that at 52 weeks, the high-
dose arm had significantly more study subjects with com-
plete/partial response in contrast to the low dose [27 (73%)
vs low dose 19 (50%), p = 0.04]. Even the non-responders
were significantly higher in the low dose [19 (50%),] com-
pared to the high dose [10 (27%) p = 0.04], although our
observations need further validation on long-term follow-
up of 5 years. In the ELNT study [8], renal remission was

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Table 1  The baseline Variable Low dose (n = 38) High dose (n = 37) p value
characteristics of the study
subjects Female/male 34/4 (89%/10%) 34/3 (92%/8%) 0.53
Age of SLE onset, Yrs Mean ± SD 28.5 ± 10.05 25.7 ± 10.35 0.24b
Age of onset of nephritis Mean ± SD 30.71 ± 10.04 27.24 ± 10.60 0.15b
Disease duration, months median, IQR 18 (4–36) 12 (3.5–24) 0.19c
Clinical features at the time of ­nephritisd
Fever/systemic 6 (16%) 9 (24%) 0.39
Mucocutaneous 23 (60%) 20 (54%) 0.57
Arthritis 14 (37%) 10 (27%) 0.46
Hemolytic anemia 11 (29%) 11 (30%) 0.94
Leucopeniaa 14 (34%) 5 (14%) 0.02
Thrombocytopenia 9 (24%) 7 (22%) 0.83
Pericardial effusion 2 (5%) 7 (19%) 0.08
Pleural effusion 6 (16%) 7 (19%) 0.08
Vasculitis 7 (18.4%) (16.21%) 0.80
Myositis 3 (8%) 2 (5.4) 1.00
Myocarditis 1(2.6%) 3 (8%) 0.35

CI confidence interval, IQR interquartile range, NA not applicable, OR odds ratio, SD standard deviation,
Yrs years
a
Significant p < 0.05
b
Student t test
c
Compared using non-parametric test Mann–Whitney test
d 2
χ test used

observed in 71% of the low dose and 54% of the high dose
(not statistically significant), and they concluded that there
was no difference in the treatment arms. There were signifi-
cant differences in our study compared to the ELNT with
respect to their definition of remission, end assessment at
41 months, a total of eight pulses (six monthly and two
quarterly) in the high-dose group along with inclusion of
patients with class V LN. Sabry et al. [19] at the end of
12 months concluded that both low-dose and high-dose CYC
achieves similar results without serious infections in both
regimens. We observed in the high-dose CYC arm, a higher
CR of 65% compared to other studies by El-Shafey et al.
[20] where CR was 21.74%, Chen et al. [21] (CR was 38%)
and Aspreva Lupus Management Study (ALMS) study [22]
(CR in only 8%). This was very low compared to our study,
as the ALMS study included more African-Americans with
relatively severe disease and class V LN. Also, in contrast,
to our study, they did not administer methylprednisolone
pulse therapy to all patients. Among the Asian subjects,
they achieved a response of 63.9% in high-dose CYC arm,
which was similar to our study. In our study, no response rate
observed in high-dose group was also significantly lower 10
(27%) vs low dose 19 (50%) at 52 weeks. This was lower
than a study by El-Shafey et al. [20] in the high-dose CYC
arm of 47.83% and 10% were non-responders in the com-
bination arm of NIH studies [6]. In the ELNT trial, at the
end of 41 months, 16% of those in the low-dose group and
20% in the high-dose group experienced treatment failure as
defined in their study [8]. Sabry et al. [19] observed treat-
ment failure at the end of 12 months in 5% of the low-dose
CYC group and 3.8% of the high dose and this was in the
form of doubling of proteinuria. In contrast, in a study by
Chen et al. [21], 6% of patients had no response. This may be
attributable to the Chinese population being genetically dif-
ferent and having lower positivity of anti-dsDNA antibodies
at baseline (46.2% in high-dose group) in comparison with
our study where anti-dsDNA positivity was 82%.
We observed that at the end of 24 weeks, there was no
statistically significant difference in complete response and
complete/partial response in the low dose vs high dose.
Complete response (CR) was 21 (55%) vs 21 (57%), while
the complete/partial response (CR/PR) was 23 (60%) vs
25 (68%). This was similar to the low dose CYC arm of
Rathi et al. [23] where they observed complete remission at
24 weeks in 25/50 (50%). Our results were better compared
to a recent study by Sigdel et al. [24], where in the low-dose
arm, at the end of 24 weeks, 43.9% achieved complete remis-
sion and 16 (39.0%) achieved partial remission. This may
be attributed to 500 mg CYC given monthly for 24 weeks
in their study. In a study by Ginzler et al. [25] in the high-
dose arm, 5.8% had complete remission and 21/69 patients
(30.4%) had either complete or partial remission. However,
a majority of their patients were blacks, and included those
with Class V LN. Our results were also similar to a study by

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Table 2  Baseline renal Variable Low dose (n = 38) High dose (n = 37) p ­value₴
parameters
Hypertension no. (%)a 18 (47%) 25 (67%) 0.07
Renal biopsy
Class IV no. (%) 21 (55%) 26 (70%) 0.17
Class III no. (%) 17 (44%) 11 (30%) 0.17
Activity Score 4 (2–18) 6 (1–15) 0.11€
Median IQR
Chronicity Score 1 (0–4) 1 (0–7) 0.51€
Median IQR
Crescents no. (%) 8 (21%) 14 (38%) 0.10
Serum creatinine mg/dL 0.95 (0.7–2.8) 0.90 (0.60–4.7) 1.00€
Median IQR
> 1.3 mg/dL 10 (26%) 13 (35%) 0.40
eGFR Mean ± SDb 68.66 ± 23 68.41 ± 32 0.96¥
eGFR no. (%)b
> 90 7 (18%) 9 (24%) 0.53
61–90 18 (47%) 14 (38%) 0.40
31–60 10 (26%) 8 (22%) 0.63
< 30 3 (8%) 6 (16%) 0.30
Serum albumin 2.89 ± 0.58 2.64 ± 0.54 0.06¥
g/dL Mean ± SD
Active urinary sediments no.(%) 30 (79%) 35 (95%) 0.08
uPCR gm/day 1.5 (0.04–9.0) 1.7 (0.60–13) 0.43€
Median IQR
< 2 no. (%) 25 (66%) 26 (70%) 0.67
2-2.9 no. (%) 3 (8%) 6 (16%) 0.30
> 3 no. (%) 9 (24%) 4 (11%) 0.14
Renal SLEDAI 8.4 ± 3.4 9.9 ± 3.4 0.06¥
Mean ± SD
Median IQR 8 (4–12) 12 (8–12) 0.06€
Low ­complements∞ 27 (71%) 28 (76%) 0.65
C3/C4 no. (%)
Anti-Dsdna > 60 IU/ml no. (%) 29 (76%) 31 (84%) 0.41
SLEDAI Median IQR 16 (6–26) 18 (6–40) 0.07€

SD standard deviation, IQR interquartile range, OR odds ratio, CI confidence interval, RBC red blood cell,
WBC white blood cell, eGFR estimated glomerular filtration rate, SLEDAI Systemic Lupus Erythematosus
Disease Activity Index. Scoring done according to the Safety of Exogenous Estrogens in Lupus Erythema-
tosus National Assessment (SELENA) modification; hpf high power field, UPCR protein creatinine ratio
Active urinary sediments: RBC > 5 per HPF, Pus cells > 5 per HPF, Cellular casts
Significant p < 0.05
∞
Low C3 < 0.90 g/l, Low C4 < 0.10 g/l
₴ 2
χ test used-for all comparisons unless otherwise mentioned
¥
Means compared using student’s t test
€
Compared using Mann–Whitney test
a
Diastolic BP > 90 mmHg
b
ml/Min/1.73 m2

Ong et al. [26], where complete/partial remission occurred
in 13/25 patients (52%) in the high-dose IV CYC group vs
25 (68%) in our study.
At the end of 12 weeks, the patients achieving complete
response (CR) in the low dose compared to the high dose
were significantly higher 18(47%) vs. 9(24%) (p = 0.03).
However, initially, the two weekly CYC administrations
were more effective than high dose. This was in spite of
the fact that the cumulative dose of CYC received by both
groups at this time point of 12 weeks was similar [median

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Table 3  Primary Response Response parameters Low dose (n = 38) High dose (n = 37) p ­value₴
outcome at 52 weeks
Complete response(CR) 17 (44%) 24 (65%) 0.08
Complete/partial response (CR/PR) 19 (50%) 27 (73%) 0.04
No response (NR) 19 (50%) 10 (27%) 0.04
₴ 2
χ test used, p significant < 0.05

Table 4  Outcome of renal Variables Low dose (n = 38) High dose (n = 37) p ­value₴
parameters at 52 weeks
Creatinine g/dl 0.90 (0.7–2.2) 0.80 (0.6–4.7) 0.52€
Median, IQR
> 1.3 g/dl no. (%) 6 (16%) 8 (21%) 0.51
eGFR Mean ± SDa 75.6 + 21.1 79.4 + 26.6 0.49¥
eGFR no. (%)
> 90 11 (29%) 16 (43%) 0.19
60–90 18 (47%) 15 (40%) 0.55
30–60 8 (21%) 3 (8%) 0.13
< 30 1 (3%) 3 (8%) 0.35
Albumin g/dL Mean ± SD 3.53 + 0.66 3.49 + 0.68 0.82¥
Active Urinary sediments no.(%) 14 (37%) 9 (24%) 0.24
uPCR gm/day 0.20 (0.01–9.0) 0.12 (0.02–4.1) 0.99€
Median IQR
< 2 no. (%) 33 (87%) 32 (86%) 1.00
2–2.9 no. (%) 2 (5.3%) 3 (8%) 0.67
> 3 no. (%) 3 (8%) 2 (5%) 1.00
Renal SLEDAI 4 (0–16) 0.00 (0–16) 0.16€
Median IQR
dsDna antibody > 60 IU/ml no. (%) 18 (47%) 15 (40%) 0.55
%Change in the dsDNA positivity 11 (29%) 16 (44%) 0.19
Low complements (no %)∞ 15 (39%) 9 (24%) 0.16
C3/C4
SLEDAI Median IQR 5 (0–26) 2 (0–30) 0.18€
Delta SLEDAI median ­IQRb 10 (5.5–14) 16 (7–20) 0.04€

IQR interquartile range, SD standard deviation, RBC red blood cell, WBC white blood cell, dsDNA Double-
stranded DNA, eGFR estimated Glomerular filtration rate, PCR protein creatinine ratio, SLEDAI Systemic
Lupus Erythematosus Disease Activity Index; Scoring done according to the Safety of Exogenous Estro-
gens in Lupus Erythematosus National Assessment (SELENA) modification; Active urinary sediments:
RBC > 5 per hpf, Pus cells > 5 per hpf, Cellular casts
Significant p < 0.05
∞
Low C3 < 0.90 g/l, Low C4 < 0.10 g/l
₴ 2
χ test-for all comparisons unless otherwise mentioned
€
Mann–Whitney U test used
¥
Means compared using student’s t test
a
ml/Min/1.73 m2
b
Difference from baseline to 12 weeks

with IQR in the low-dose group 3 (3–3) gram vs 3 (2.7–3)
in the high-dose group, p value 0.8]. The presence of higher
complete response in the low dose may be attributable partly
to the difference in the mean time to recovery of circulat-
ing granulocytes and lymphocytes that is 3 weeks and the
effects on their circulating leukocyte precursors and their
subpopulations [27, 28]. Our observations were higher than
reported in another recent study [23], in which at the end
of 12 weeks in the low-dose regimen, renal remission was
observed in 16/50 study subjects (32%). Our results may be

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Rheumatology International

Table 5  Adverse events during Adverse events Low dose (n = 38) High dose
the induction therapy (n = 37)

No. of deaths 2 (5%) 2 (5%)

Benign Non Renal flare 7 (18%) 3 (8%)
Severe Non Renal F ­ larea 1 (3%) 1 (3%)
d
Renal ­Relapse 9 (24%) 1 (3%)
Time to relapse weeks (Median IQR) 48 (41–50) 46 (40–52)
Steroid-induced DM 3 (8%) 1 (3%)
Steroid-induced acne 4 (10%) 4 (11%)
Alopeciab 2 (5%) 8 (22%)
Gastrointestinal (nausea/vomiting/diarrhea) 0 (0%) 4 (9%)
Infections 3 (8%) 2 (5%)
Total no. of episodes 20 (53%) 17 (46%)
Severe infections
Sepsis 3 (8%) 2 (5%)
Pneumonia 4 (10%) 2 (5%)
Osteomyelitis 1 (3%) 0 (0%)
URTI/nasopharyngitis 5 (13%) 7 (19%)
Herpes Zoster 1 (3%) 0 (0%)
Varicella Zoster 0 (0%) 1 (3%)
Urinary tract infection 1 (3%) 0 (0%)
Strongyloides infection 0 (0%) 2 (5%)
Oral candidiasis 5 (13%) 3 (8%)
Skin tissue infections 4 (10%) 4 (11%)
Cutaneous Pheaohyphomycosis 0 (0%) 1 (3%)
Toxic anemia 2 (5%) 6 (16%)
New onset CYC-induced Leukopenia (< 4000/cumm)c 0 (0%) 5 (13%)
New onset Azathioprine-induced Leucopenia (< 4000/cumm) 6 (16%) 3 (8%)
Thrombocytopenia 0 (0%) 1 (3%)
Transaminitis 1 (3%) 5 (13%)
Menstrual irregularity (temporary) 6 (16%) 9 (24%)
Menstrual irregularity (permanent) 1 (2%) 2 (5%)
New onset avascular necrosis 3 (8%) 1 (3%)

DM diabetes Mellitus, URTI upper respiratory tract infection, CYC​cyclophosphamide

a
Gastrointestinal vasculitis in low dose and Thrombotic thrombocytopenic purpura in the high dose
b
p, OR 95% CI 0.04 [4.9 (0.97–25.2)]
c
p OR 95% CI 0.01 [1.1(1.08–1.3)] χ2 test,
d
p, OR 95% CI 0.01 [0.09 (0.01–0.74)]

explained additionally by the findings of Mc Cune et al. [29],
in which they studied the reduction in the B lymphocyte
and antibodies as well the T-cell repertoire during monthly
cyclophosphamide therapy, and this reduction may be more
efficient during two weekly regimen. A similar study by
Martin-Suarez et al. [30] studied that weekly administra-
tion of the low dose of CYC in multiple connective tissue
disorders including LN was observed to be beneficial in
inducing an early response in LN. Interestingly, Houssiau
et al. [31] studied that those patients that had a good long-
term renal outcome in the ELNT trial had an earlier and
significant reduction in the serum creatinine levels, serum
albumin levels, and proteinuria during the first 6 months of
therapy. However in our study, the interesting observation
of an early response in the low dose that was not sustained
until 52 weeks. In fact, the renal relapses were higher in the
low dose.
The baseline SLEDAI (median, IQR) in the low dose
was 16 (6–26) and high dose was 18 (6–40), and they were
reduced to 5 (0–26) in the low-dose and 2 (0–30) the high-
dose arm, with no intergroup difference. The SLEDAI score
at baseline was comparable to other studies [21–23, 32]. At
52 weeks the change in the SLEDAI, defined as delta SLE-
DAI (median, IQR) was significantly higher in the high dose

13

16 (7–20) vs low dose 10 (5.5–14) [p = 0.04]. In the ELNT
study [8], disease activity was assessed by ECLAM score,
and in contrast to our study, they observed no difference in
treatment arms. The percentage of patients with anti-dsDNA
antibody positivity were 76 and 84%, similar to other studies
[22, 23]. The decline in percentage positivity of anti-dsDNA
antibody was 29% in low dose and 44% of the high dose.
The overall adverse events profiles of both low-dose
and high-dose CYC were consistent with previous studies
[8, 19, 25]. The mortality rate in our study was similar to
Rathi et al. [23], 4% in the low-dose CYC group and 2.8%
in the high-dose CYC arm of the ALMS study [22]. The
mortality rate was similar in both the groups, however,
in the ELNT trial, there was no mortality in the high-
dose regimen. For both the treatments, the most common
adverse events were infections, and similar to the ELNT
trial, there was no difference statistically significant differ-
ence between the groups. Alopecia occurred significantly
in the high-dose arm of CYC (p = 0.04), which was simi-
lar to other studies [3, 21]. Leucopenia was more in the
high-dose CYC arm (13%), which was not observed in the
low-dose group (p = 0.02). However, this did not lead to
discontinuation of therapy. This was lower than reported
by Ginzler et al. [25] in their high-dose CYC arm of 37%.
Azathioprine-induced leucopenia occurred in 16% of the
low dose that was similar to the low-dose arm of ELNT
trial [8] and in the MAINTAIN trial by Contreras et al.
[33]. There was no difference in the development of men-
strual irregularity between the two treatment arms (16% of
the low dose and 24% at the high dose). Premature ovarian
failure was observed in 20% in the CYC arm by Ginzler
et al. [25], and Boumpas et al. [7] observed premature
ovarian failure in 19% in the modified NIH arm. This was
in contrast to the ELNT trial, where they observed sig-
nificantly lower gonadal toxicity in Caucasians (6.8% in
the low dose arm and 6.6% in the high dose arm) [8].
This may be attributable to genetic differences. This was
much lower than that observed in the standard NIH regi-
men where the gonadal failure rate was higher, varying
from 38%-52% [7]. During the study period, none of our
study subjects developed doubling of serum creatinine and
only one in the high-dose group developed end-stage renal
disease requiring dialysis. The benign non-renal flares
were predominantly mucocutaneous, and occurred more
in low-dose arm compared to the high-dose arm (18 vs.
8%). Severe extra renal flare of gastrointestinal vasculitis
occurred in one study subject in the low-dose arm during
induction and one patient in the high-dose arm developed
thrombotic thrombocytopenic purpura during maintenance
therapy with azathioprine. The renal relapses were sig-
nificantly higher in the low-dose vs high-dose group [9
(24%) vs 1(3%), p = 0.01]. In the ELNT trial [8], renal
flares were noted in 27% of the low-dose group and 29%
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Rheumatology International
of the high-dose group. This difference may be explained
by a longer duration of follow up of 41 months.
Limitations
This was a single center study with subjects of single eth-
nicity and needs validation in a larger multicenter cohort.
The study was not blinded. However, this probably repre-
sents a more real-life clinical scenario than blinded clini-
cal trials. Outcomes such as premature gonadal failure and
malignancy need longer follow up.
Conclusion
At the completion of 52 weeks, the high-dose CYC therapy
is more effective in inducing remission in Indian patients
with LN. The disease activity was significantly lower with
decreased renal relapses and non-renal flares in the high-
dose group. Analyzing the kinetics of response, even the
predictors of renal flare such as hypocomplementemia and
high dsDNA titres reflect better efficacy of high-dose regi-
men in our population without increasing the infection risk.
The mortality rates and adverse events were also compara-
ble. However, long-term follow-up might be needed with a
larger sample size for analyzing sustained renal responses
and estimating the renal relapses. The potential implica-
tions of our study are that in Indian patients with prolif-
erative lupus nephritis, a higher dose of cyclophosphamide
was more effective in achieving a complete response with
reduced relapses at the end of one year. Therefore, in our
population, the low-dose ELNT regimen for administration
of cyclophosphamide may not be as effective as in other
populations [5, 8]. Also, the use of high-dose cyclophospha-
mide had similar adverse events compared to the low-dose
group. However, our findings need confirmation on long-
term follow-up. Further studies investigating the kinetics of
response to two weekly CYC administrations compared to
monthly for an earlier response to induction of complete/
partial renal responses compared to monthly that showed a
delayed response to induction.
Acknowledgements We thank the Indian rheumatology association for
providing us funding for this project on lupus nephritis.
Funding This study was funded by the Indian rheumatology associa-
tion (Research Grant number 2013)
Compliance with ethical standards
Conflict of interest Sonal Mehra declares that she has no conflict of
interest. Jignesh Usdadiya declares that he has no conflict of inter-
Rheumatology International

est. Vikramraj Jain declares that he has no conflict of interest. Durga 13. Hochberg MC (1997) Updating the American College of Rheu-
Prasanna Misra declares that he has no conflict of interest. Vir Singh matology revised criteria for the classification of systemic lupus
Negi declares that he has no conflict of interest. erythematosus. Arthritis Rheum 40(9):1725
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harlal Institute of post graduate medical education and research com- 15(2):241–250
mittee Number : JIP/IEC/SC/2013/5/435 dated 4.3.2014 and with the 15. Morel-Maroger L, Mery JP, Droz D, Godin M, Verroust P, Kourilsky
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ethical standards renal biopsies. Adv Nephrol Necker Hosp 6:79–118
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participants included in the study. and European Renal Association- European Dialysis and Transplant
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